In a barrage of tweets fired off during Mr Trump's final clash with his Democratic challenger, Joe Biden, Ms Trump lashed out at her uncle's handling of the coronavirus pandemic, his business debts and "good" relationship with North Korea dictator, Kim Jong-Un.
With tens of millions of Americans watching on as the two presidential candidates set out their competing visions for the country, Ms Trump, 55, berated her uncle's "evil" and "incompetent" administration.
"The only safe place for Donald to live is the past," said Ms Trump, who recently published a tell-all book about her uncle. "He will never take responsibility for anything. Ever."
"The only thing more grotesque than the cruelty and the lying is the racism," Ms Trump added, as her uncle was quizzed by NBC debate moderator, Kristen Welker.
Ms Trump, a psychologist, businesswoman and author, is the daughter of Fred Trump Jr, Donald's older brother.
Ms Trump was thrust into the international spotlight in July this year following the publication of 'Too Much and Never Enough,' a bombshell exposé detailing how the Trump family “created the world’s most dangerous man”.
Estranged from her uncle for years, Ms Trump was the first member of the family to break ranks and criticise the president, who she said has severe mental health issues and is not fit to lead the US.
Ms Trump also alleged in the book that the president's sister, a future federal judge, did his college homework for him and that he cheated on his university SAT entrance exams.
Since the publication of the book, which sold almost 1 million copies on the day it hit the shelves, Ms Trump has been a vocal critic of the president, 74, and recently came out in support of Joe Biden, 77.
In September, Ms Trump filed a lawsuit claiming the president cheated her out of tens of millions of dollars while pushing her out of the family’s real estate business.
Seeking unspecified damages, Ms Trump alleged in a New York City state court that the president, along with his brother, Robert, and sister, Maryanne Trump Barry, secretly took her share of the family’s real estate holdings while claiming to be her protectors.
“Fraud was not just the family business — it was a way of life,” the lawsuit said.