Tilikum, thought to be 36, had faced serious health issues including a persistent and complicated bacterial lung infection, park officials said.
A post-mortem examination will be carried out to establish his cause of death.
Tilikum, who was over 22ft long and weighed 11,800lbs, was the subject of the documentary Blackfish which argued killer whales, when in captivity, become more aggressive toward humans and each other.
After it was aired, animal rights activists increased their demonstrations outside Seaworld parks, crowd numbers fell and the company faced falling profits.
In 2010, SeaWorld Orlando trainer Dawn Brancheau was interacting with Tilikum towards the end of a show when he pulled her from a platform by her arm and held her under water.
She drowned but also suffered severe trauma, including multiple fractures.
Tilikum was also involved in previous controversies, including in 1999 when a naked man who sneaked into SeaWorld at night was found dead the next morning draped over the orca in a tank.
And in 1992 at Sealand of the Pacific in Canada, Tilikum and two female orcas were responsible for the death of a part-time trainer who slipped and fell into their pool and was submerged by them.
In March last year, SeaWorld chief executive Joel Manby admitted the public’s attitude had changed about keeping killer whales and the company would end its orca breeding programme.
“We needed to move where society was moving,” Mr Manby said.
Reacting to his death, he said: “Tilikum had, and will continue to have, a special place in the hearts of the SeaWorld family, as well as the millions of people all over the world that he inspired.
“My heart goes out to our team who cared for him like family.”
Tilikum was SeaWorld’s most prolific male orca, fathering 14 calves while he was at the Orlando park after arriving about 25 years ago.