In a statement to ABC News, Ms Giuffre said she was suing Andrew after allegedly being sex trafficked to him and sexually abused by him.
"I am holding Prince Andrew accountable for what he did to me," she said.
"The powerful and rich are not exempt from being held responsible for their actions […] I did not come to this decision lightly.
"As a mother and a wife, my family comes first — and I know that this action will subject me to further attacks by Prince Andrew and his surrogates — but I knew if I did not pursue this action, I would be letting them and victims everywhere down."
Prince Andrew has denied all of Ms Giuffre's claims and a representative for the royal said there will be no comment on the lawsuit.
In 2019, the Duke of York told BBC Newsnight that the alleged sexual assault "didn't happen" and that he had "no recollection" of ever meeting Ms Giuffre.
"I can absolutely categorically tell you it never happened," he said at the time.
Ms Giuffre alleges that she was sexually abused by Andrew in 2001, her claims first coming to light in 2015. Buckingham Palace denied the allegations.
WATCH: Prince Andrew's accuser Virginia Giuffre is interviewed by the BBC. Story continues below.
She reiterated her allegations in a 2019 BBC interview, at which time Buckingham Palace denied her claims once again.
"It is emphatically denied that The Duke of York had any form of sexual contact or relationship with Virginia Roberts [Giuffre]," the palace said in a statement.
"Any claim to the contrary is false and without foundation."
Prince Andrew faced criticism for his ties to Jeffrey Epstein, who died in 2019, and stepped back from royal duties that same year.