Judy Zagorski, 57, was on holiday with her family on the boat when the bizarre incident happened.
It was not immediately clear if she was killed by the barb of the 6ft spotted eagle ray or whether she suffered a fatal injury when she banged her head.
The incident off the Florida Keys was reminiscent of the freak death of Australian wildlife expert Steve Irwin who was killed by a different type of ray while filming on Australia's Great Barrier Reef in 2006 when its barb pierced his heart.
Spotted eagle rays can grow up to 17 feet in length and weigh up to 35 stone. They are known to occasionally jump out of the water, but are not aggressive.
"It's a bizarre accident," said Jorge Pino, an agent with the Florida Fish and Wildlife Conservation Commission.
Mrs Zagorski and her familym from Michigan on an early Easter break, were aboard a boat in the Atlantic Ocean, off the city of Marathon in the Florida Keys, he said.
"A large ray jumped out of the water and collided with the victim and somehow the barb penetrated some part of her body, which caused her to fall back and hit her head on some portion of the vessel," Pino said.
"We don't know exactly which one of those things caused her death."
Her father who was driving the boat " had absolutely no warning. It just happened instantaneously," Pino said.
Eagle rays are common in warm or tropical waters and are often seen near coral reefs. The spotted creatures can have two to six short, venomous barbs near the base of their whip-like tails.
The rays often swim near the water's surface and can leap out, especially when pursued, but are generally shy of humans.
"All rays leap out of the water from time to time but certainly to see one collide with a vessel is extremely unusual," Pino said.
"Rays jump to escape a predator, give birth and shake off parasites," said Lynn Gear, supervisor of fishes and reptiles at Theater of the Sea in Islamorada.
"They do not attack people."
