He explained the organisation which bears his name was non-partisan, before making the joke about Michelle Obama.
"I've held my last political office," the 57-year-old said. "Michelle would leave me if I ever ran for office again."
He was greeted by a smattering of laughter from the audience, while Mr Obama continued talking with a straight face.
Barack Obama's best moments in the White House
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The 44th US president went on to say that he remained "deeply invested" in issues including tolerance, equality, rule of law, democracy and human rights.
While he ruled out a return his wife has been questioned on several occasions regarding whether she would ever consider running to become US president.
During the promotional tour for her memoir Becoming, the lawyer was asked by television talk show host Jimmy Kimmel whether she's been "seriously approached" by those wishing her to take up the mantle.
"No, I haven't had any serious conversations with anyone about it because it's not something that I'm interested in or would ever do. Ever," the 55-year-old replied.
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Last November, Mr Obama made a surprise appearance during a stop on Michelle's book tour in Washington, DC.
Obama arrived on stage with a bouquet of flowers for his wife, much to the excitement of the audience.
"You know when Jay-Z comes out during a Beyoncé concert?" he joked.
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