The focus on Rubio offers a potential preview of Trump's strategy for the fourth Republican presidential debate
"Marco Rubio is a total lightweight who I wouldn't hire to run one of my smaller companies -- a highly overrated politician!" Trump tweeted.
Trump followed that tweet with criticism of how Rubio delivered the Republican response to Obama's State of the Union address in 2013.
"Marco Rubio couldn't even respond properly to President Obama's State of the Union Speech without pouring sweat & chugging water. He choked!"
He combined both messages in his final tweet of the evening on the subject: "Marco Rubio is totally weak on illegal immigration & in favor of easy amnesty. A lightweight choker -- bad for #USA!"
The twitter attack matched Trump's language earlier in the day at a packed campaign rally in Illinois.
At the event, the business mogul said of Rubio, "I look at a guy like Rubio, who's very, very weak on illegal immigration. He was a member of the Gang of eight -- that meant 'come in.'"
While the billionaire businessman has managed to stay in first or second place in most recent polls -- behind only neurosurgeon Ben Carson -- Rubio has risen in recent days to third place in many surveys, buoyed in part by a positive performance at the last debate last month.
Trump also appeared on ABC's "Good Morning America" on Tuesday and offered only a coy preview of the night's debate.
Asked by host George Stephanopoulos whether he would "tee off" on Carson, Trump demurred, saying "No not at all, we'll see what happens. It will be loose, it will be very flexible. I have no idea what's going to happen, but it will be interesting."