The first weekly Tui flight to Orlando from Doncaster Sheffield airport took off on Saturday (4 May).
Celebrations at the airport included a Disney-themed party in departures.
Tui says the new route is part of its aim to ensure more holidaymakers can jet off from their local airport.
According to the operator, its Doncaster store has sold more holidays to Florida than any other in the country.
It comes after Tui announced flights to Cancun in Mexico from Doncaster Sheffield, starting summer 2020.
In addition, the operator has also unveiled a new route to Girona in Spain, serving Barcelona, from Doncaster Sheffield, also taking off for summer 2020.
Dawn Wilson, managing director Tui Airways, said: “We are so excited to be launching flights from Doncaster Sheffield to Florida, as we know how popular Florida is for UK holidaymakers and the importance of flying from a local airport.
“We are also delighted to be announcing a brand-new summer 2020 route from Doncaster Sheffield to Girona, a popular Spanish destination which will operate alongside a long-haul favourite in Cancun, Mexico. We are confident customers will benefit from our continued growth at Doncaster Sheffield airport.”
The Orlando flight was also Doncaster Sheffield’s maiden long-haul flight. Chris Harcombe, the airport’s aviation development director, said it was a “momentous day” for Doncaster Sheffield.