"I have concluded that, if the Senate issues a subpoena for my testimony, I am prepared to testify," he said.
Senate Majority Leader Mitch McConnell has publicly expressed resistance to calling new witnesses in the trial, though Democrats are pressing to hear from Bolton and others who did not appear before the House's inquiry in the upcoming proceedings.
Bolton's statement comes as House Speaker Nancy Pelosi is stalling House-passed articles of impeachment against Trump in a bid to get new witnesses to testify. Senate Democratic Leader Chuck Schumer has proposed calling several witnesses, including Bolton, but McConnell has so far rejected Schumer's terms.
Bolton served from April 2018 to September as Trump's third national security adviser. He was ousted over disagreements with Trump over how to deal with Iran, Afghanistan and a host of global challenges. Trump later named attorney and former ambassador Robert C. O'Brien to serve as his fourth national security adviser.
Bolton had been asked to testify last year as part of the House impeachment inquiry but he and his former deputy, Charles Kupperman, declined and asked a court to clarify whether they were legally required to appear. Trump had earlier ordered White House officials not to cooperate with the inquiry, which he framed as illegitimate.
Democrats launched the impeachment inquiry after learning of a whistleblower complaint filed in August by an official concerned over Trump's attempts to get Ukrainian officials to investigate former vice president and 2020 Democratic presidential hopeful Joe Biden.
Trump has denied any wrongdoing.
In a closed-door interview with lawmakers in October, Fiona Hill, a former White House adviser on Russia, testified Bolton was so disquieted by back-channel Ukraine activities that he referred to Trump's personal attorney, Rudy Giuliani, as a "hand grenade who is going to blow everybody up."
The Associated Press contributed to this report.