In a unanimous vote, the newly-elected legislative body fired Ortega. In addition to barring her from ever seeking public office again in Venezuela, lawmakers prohibited her from leaving the country and froze her assets.
In a live broadcast on state TV, the assembly's president said Ortega would be removed and legal action would be brought against her. The news was met with applause by the more than 500 members of the legislative body, known as the Constituents.
The National Constituent Assembly is expected eventually to take the place of the opposition-led National Assembly in a move that critics fear will erode democracy.
The new assembly, which has wide-ranging powers and is expected to rewrite the Venezuelan Constitution at the behest of leftist President Nicolas Maduro, had its first session televised live on state television.
Just before Ortega was fired, government troops prevented her and some of her subordinates from entering her Caracas office building.
More than three dozen troops stood outside the building Saturday, pictures released by her press office show.
"This is a dictatorship -- the abuse that is being experienced in Venezuela, the repression," Ortega told reporters there.
The firing defied the regional Organization of American States, whose human rights commission Saturday warned the Venezuelan government to guarantee Ortega's safety and allow her to continue as attorney general.
It cited what it said were comments by "high-ranking officials" indicating Ortega may face prosecution by Maduro's regime.
With the ouster, Ortega has lost immunity from prosecution that Venezuelan government officials enjoy. Venezuela's Supreme Court issued a statement that she will be investigated over what it called serious violations and offenses as attorney general.
The new attorney general's office
The assembly proposed a "restructuring" of the attorney general's office and nominated Tarek William Saab
, a Maduro ally and former ombudsman, to be the interim attorney general.
He was sworn in late Saturday afternoon to a rousing applause from the Constituent Assembly.
During his swearing-in speech, Saab blamed the inaction of the attorney general's office for the wave of violence that has gripped the country.
"The attorney general's office allowed for there to be an approximate number of 100 victims for the lack of justice, for not acting early on, for allowing violent groups to gain unworthy control of territories that need to be liberated for the peace of all citizens."
The US Treasury Department issued sanctions in July against Saab and 12 other Maduro loyalists.
"As President Trump has made clear, the United States will not ignore the Maduro regime's ongoing efforts to undermine democracy, freedom, and the rule of law," Treasury Secretary Steven Mnuchin said at the time.
"As our sanctions demonstrate, the United States is standing by the Venezuelan people in their quest to restore their country to a full and prosperous democracy. Anyone elected to the National Constituent Assembly should know that their role in undermining democratic processes and institutions in Venezuela could expose them to potential US sanctions."
Saab has also been openly criticized by many people around the country, including his son Yibram, who condemned the deaths of young protesters on a YouTube video that went viral.
Ortega had been a target in recent months
Once a staunch supporter of the late Venezuelan President Hugo Chavez's leftist movement, Ortega recently became a critic of the government under Maduro, Chavez's handpicked successor.
In recent months she has spoken out against the Venezuelan national guard and the Maduro government over human rights abuses committed against protesters. She has also opposed the National Constituent Assembly.
For months, Ortega has denounced the Venezuelan National Guard and the National Police for using excessive force against protesters. She has been at odds with the government, accusing high-ranking current and former members of the country's law enforcement branches of committing human rights abuses.
Ortega had also recently accused Maduro's government of committing "state terrorism" by stripping citizens of their right to protest, trying them in military courts and carrying out raids without consulting courts.
"We continue to witness the rupture of the constitutional order. The constitution keeps on being violated, and the government institutions are being dismantled," she said in June.
Hours after being fired, Ortega issued a statement saying that the work by the Constituent Assembly is a coup on the constitution, and she vowed to not surrender to the government.
"I will continue to fight for all Venezuelans, for their liberties and their rights, until my last breath and in whatever position I might be in."
Months of strife
Venezuela has struggled with a collapsing economy for many years. Maduro's opponents attempted to hold a referendum vote to remove him from office after they won a National Assembly majority in 2015, but the Supreme Court, stacked with government supporters, blocked all attempts to remove him.
Maduro has also delayed local and state elections.
The National Constituent Assembly's election follows months of sometimes deadly anti-regime protests and an economic crisis
that led many to leave Venezuela
in search of easier access to food and medicine. This latest wave of unrest started after the Supreme Court of Venezuela attempted to dissolve the opposition-led National Assembly.
For his part, Maduro has said the election of this constituent body was needed to help establish order and peace in the country.
His opponents, meanwhile, say the new assembly could erase the last traces of democracy in the South American country. Opposition leaders say it was a last-ditch effort by Maduro to get rid of the opposition and the checks and balances between the branches of government.
The opposition-led National Assembly has indicated it will continue meeting -- raising prospects that two camps may claim to be the country's legitimate government
Saturday, South America's trade bloc, Mercosur, decided to suspend Venezuela indefinitely from the group until there was a "re-establishment of democratic order." The Organization of American States applauded that decision.
Mercosur's decision is mostly symbolic and will not impact trade or the free movement of Venezuelans among Mercosur countries.
Brazil's state-run news agency quoted Brazilian Foreign Minister Aloysio Nunes Ferreira as saying that the Mercosur decision is of a "serious political nature," but that they took into consideration "the concern of not creating greater problems for the Venezuelan people. Venezuelans who want to come to Brazil will be welcomed."Both Maduro and Venezuela's Foreign Minister Jorge Arreaza rejected the decision by the block.
US State Department spokeswoman Heather Nauert said this week that the United States won't recognize the National Constituent Assembly because "the process was rigged from the start" and "the election lacked credible international observation."
Assembly's next steps
Saturday's session was a busy one for the newly established body. Not only did members restructure the attorney general's office, but they also unanimously approved extending to two years the time the assembly will stay in power. Currently, the constitution allows for only six months.
Assembly leader Delcy Rodriguez, a former Venezuelan foreign minister, warned Friday that "justice will come"
to some members of the opposition. Saturday, she announced that the Constitutional Assembly would create a "truth, justice and peace commission" to investigate the violence in the country in the past few months.
Minutes after announcing the commission, Rodriguez also attacked Ortega, saying she had used her office to destabilize the country.
"She opened the door for foreign interference in Venezuela. She also opened the door for the fascist right to act with impunity, assassinating and bringing violence to our youth."
Rodriguez plans to lead the truth, justice and peace commission in its first meeting Sunday.
Then, on Tuesday, the full assembly is expected to convene for its second meeting.