"There is no sex tape," she said in a statement to People on Tuesday. "There never has been one and there never will be one."
Until now, Bullock, 45, hasn't spoken of the matter and stayed out of the public eye.
James and his camp have said little since a woman told In Touch magazine that she carried on an affair with James while Bullock was shooting the blockbuster hit "The Blind Side."
The film earned Bullock widespread acclaim that culminated with a best actress Oscar. Within days of her win, the cheating allegations broke and Bullock withdrew from the public eye.
James released a statement saying he had used "poor judgment" and deserved "everything bad that is coming my way."
Public support for the actress has remained high as more women have stepped forward claiming they had affairs with James in recent years.
Bullock and James met in 2003 and were married in 2005. It seemed like an odd pairing - the Hollywood actress often called "America's Sweetheart" marrying the tattooed biker who designed custom motorcycles and starred in reality shows such as "Monster Garage" and "Jesse James is a Dead Man."