The former mayor of New York responded on Twitter after the movie screenshot showing him lying on bed in front of an actress went viral online.
“The Borat video is a complete fabrication. I was tucking in my shirt after taking off the recording equipment,” Mr Giuliani said.
“At no time before, during, or after the interview was I ever inappropriate. If Sacha Baron Cohen implies otherwise he is a stone-cold liar.”
Mr Giuliani, 76, was captured on hidden cameras set up in a hotel suite after actress Maria Bakalova, playing a fake conservative TV show host, invited him up to her room for a “drink”.
Her fictional father Borat, a Kazakh journalist played by Baron Cohen, busts into the room shouting: “She’s 15. She’s too old for you” as part of the prank in Borat Subsequent Moviefilm, which will be released on Friday on Amazon Prime Video.
The film scene first came to light in July after Mr Giuliani reported it to police.
In his tweet thread in defence of the scene, Mr Giuliani said it was an effort to blunt his “relentless exposure of the criminality and depravity of Joe Biden and his entire family”.
“We are preparing much bigger dumps off of the hard drive from hell, of which Joe Biden will be unable to defend or hide from. I have the receipts,” he said.
Joe Biden called Mr Giuliani a “henchman” of Donald Trump and said he was leading a desperate, last-ditch smear campaign against the Biden family.
Mr Giuliani is linked to a controversial story in the New York Post suggesting the former vice president was inappropriately connected to his son Hunter’s former job as a member of the board of a Ukrainian energy company called Burisma.
Mr Trump was impeached by Congress last year for trying to pressure the Ukrainian president to help dig up dirt on Mr Biden, a scheme Mr Giuliani was closely involved in.