The New York senator, one of many candidates to go after the front-runner, seized on an op-ed Biden wrote in 1981 about the "deterioration of the family."
"What did you mean when you said when a woman works outside the home it's resulting in 'the deterioration of family'?" she asked, suggesting Biden was not supportive of working mothers.
According to an NPR fact-check of the exchange, Biden's op-ed was an explanation of why he opposed giving a child care tax credit to people with higher incomes.
He wrote that, "A recent act of Congress puts the federal government in the position, through the tax codes, of subsidizing the deterioration of the family. That is tragic."
Biden reminded Gillibrand of her past support and recalled his time as a single father after his first wife and the couple's 1-year-old daughter were killed in a car accident in 1972.
"As a single father who, in fact, raised three children for five years by myself, I have some idea what it costs," he shot back, reminding Gillibrand that she once traveled to Syracuse University with him and applauded him for his work on behalf of women's equality.
"I don't know what's happened, except that you're now running for president," Biden told Gillibrand.
His campaign hit back on Twitter after the debate, saying the attack from Gillibrand was "two weeks in the making" and inaccurate.
Lee Carter of Maslansky Partners, who analyzed voters' real-time reactions to the moment, said on "Fox & Friends" that the challenge from Gillibrand fell flat.
"I think this was Gillibrand's big moment and it went bust," she said, explaining that it failed to move the needle with Democrats, Republicans and independents on her voter dials.