"Let me be clear: I do not want, nor will I accept the nomination for our party," Ryan said in remarks from the Republican National Committee's headquarters in Washington.
In a plea to delegates, Ryan added: "I believe you should only choose from a person who has actually participated in the primary. Count me out."
With his conservative credentials and appeal to the party establishment, a growing number of Republicans had publicly and privately floated Ryan as the lone candidate who could unite a party in disarray as it heads into a possible contested convention in Cleveland.
But Ryan, who also has the job as chairman of the convention, wanted to dispel any notions that he's plotting his own presidential bid. Behind the scenes, Ryan is making clear that his biggest goal in 2016 is now helping save an increasingly endangered House GOP majority. Ryan now will begin a furious fundraising push for his colleagues and to bolster his own coffers. According to a list obtained by CNN, Ryan will hold nearly 20 fundraising events over the next eight weeks, capping off with a June money event in Hot Springs, Virginia.
While Ryan has repeatedly said he's not interested in a White House run, many GOP supporters argued he made similar comments when he initially said he would never become the speaker of the House.
On Tuesday, Ryan dismissed comparisons between speculation that he would accept the speakership -- which he repeatedly denied before taking the position last fall -- and his refusal on Tuesday to be considered for the GOP's nomination at the Republican convention in July. He suggested convention delegates should adopt a rule only allowing a candidate who has run to be the party's presidential nominee.
Asked by CNN if he believes only one of the final three 2016 Republican candidates -- Donald Trump, Texas Sen. Ted Cruz and Ohio Gov. John Kasich -- should be the nominee, Ryan would only say, "I'll leave that up to the delegates."
"We should be selecting among people who actually ran for the job," Ryan added.
If delegates follow his advice, Ryan's position means other former candidates -- like Florida Sen. Marco Rubio, Wisconsin Gov. Scott Walker or former Florida Gov. Jeb Bush -- could find themselves in the fold if the convention is deadlocked.
Minutes before Ryan was scheduled to address reporters in the lobby of the RNC, dozens of protestors visiting Washington for a "Democracy Spring" gathered outside. The group is advocating rules to remove the influence of money in politics and loud chants of "free and fair elections" were heard inside the room where about a dozen cameras and throngs of reporters gathered to cover the speaker's remarks.
The move comes as the chatter over a possible Ryan bid at a deadlocked convention is becoming a major distraction for the speaker. But Ryan aides believe even a late entry would not have been feasible and would have divided the party even further.
Ryan continues to stress that his main priority is developing an agenda for the eventual nominee to adopt, to show what Republicans would do to replace Obamacare, reform the tax code, address poverty and boost job creation.
Even before Ryan planned to address the issue on Tuesday, conservatives were warning that any move to install him as the party's standard-bearer at the Republican convention in Cleveland would backfire.
"The one thing that would completely destroy the party is coming out (of Cleveland) with Paul Ryan as the nominee," Republican South Carolina Rep. Mick Mulvaney told CNN on Monday.
Another conservative told CNN he opposed Ryan -- or any other individual who hasn't been campaigning -- from getting the party's nomination.
"To parachute someone in who hasn't run for the office, who hasn't done the grueling work of campaigning -- if you want to do something that heightens cynicism to all-time high, that would do it," Virginia Rep. Dave Brat told CNN on Monday.
Several House Republicans told CNN that Ryan has been clear with them that he has been trying to tamp down the speculation and he doesn't want to run for president. They say he made a conscious decision when he decided to run for speaker last fall that he was removing himself from a White House bid.
Mulvaney said his concerns had nothing to do with Ryan personally, but because Republican activists would feel "something was taken from them unfairly and they would stay home." He predicted 60% of the voters in his district wouldn't vote in November if the convention handed the nomination to the House speaker.
"I think it's simply nonsense that someone outside of the three people running for this nomination are going to be nominated at this convention," Colorado Sen. Cory Gardner told CNN's John Berman on "At This Hour." "It's just nonsense. One of the three will be our nominee, and I know there are some people who think there is going to be this through-the-looking-glass-moment for Paul Ryan or perhaps others, but it's simply not going to happen."
Democratic National Committee Chairwoman Debbie Wasserman Schultz also commented on Ryan's announcement, which she said is no surprise given the contentious GOP nomination fight.
"I'm not at all surprised he doesn't want to step in the way of this trainwreck," Wasserman Schultz said.