The man unfurled the banner while standing near the stage during a curtain call for A Bronx Tale, directed by De Niro and four-time Tony Award winner Jerry Zaks.
The US President and De Niro have been involved in a public spat since the 74-year-old Hollywood actor received a standing ovation for yelling “f*** Trump” on stage at the Tony Awards in New York earlier this month.
The Raging Bull star was introducing a performance by Bruce Springsteen when he said: “First, I wanna say, f*** Trump… It’s no longer down with Trump, it’s f*** Trump.”
Addressing Springsteen, he added: “Even more important in these perilous times, you rock the vote. Always fighting for, in your own words, truth, transparency and integrity in government. Boy do we need that now.”
Trump, 72, responded on Twitter by calling the double Academy Award winner a “very low IQ individual” on Twitter.
The row took another turn on Saturday when the audience member pulled out the banner which read “Keep America Great” as well as “Trump 2020”.
Joe Del Vicario took a picture of the incident and said: “The times we live in. At the end of A Bronx Tale, a man turned around and faced the audience to show us how big of a pair he has.
“He was escorted shortly after. It’s sad that people can’t enjoy a beautiful show and embrace its unifying message without politicising it.”
The incident took place during the curtain call of the 8pm performance at the Longacre Theatre, on West 48th St, just off Broadway.
De Niro has been a vocal opponent of Trump in the past, calling him “an idiot”, a “dog” and a “national disaster” in a 2016 video.