Dutch-Belgian actress Sand Van Roy, accused Mr Besson of repeatedly raping her over a two-year period.
The case was reopened last week, eight months after prosecutors dropped it. Eight other women have accused the director of sexual misconduct.
Mr Besson dismissed Ms Van Roy as a "fantasist", although he admitted having had a relationship with her.
Speaking publicly about the claim for the first time, he told BMFTV that it was a "complete and utter lie". He added: "I have never raped a woman in my life."
Ms Van Roy accused Mr Besson of raping her at a hotel in Paris in May 2018. But the original investigation ended when Paris prosecutors said there was not enough evidence.
Besson, 60, a director, producer and screenwriter, is most famous for directing the 1988 film The Big Blue, as well as Subway, Leon, The Fifth Element and action thriller Nikita.
He recently directed the action thriller Anna, starring Sasha Luss and Helen Mirren.
Ms Van Roy, 28, played minor roles in two of his films, Taxi 5 and Valerian and the City of a Thousand Planets.