The direct connection "will boost our economic growth and bring our country closer to the United States and American people", said Serbian Prime Minister Aleksandar Vucic at the launch, also attended by US ambassador Kyle Scott.
Departing in the morning after a 40-minute delay, the flight was to due to reach New York's JFK airport in ten hours. The service is set to run five times a week.
Air Serbia is now the only regional airline offering direct flights to the United States, and a company official said 15,000 tickets have already been sold, according to state-run media.
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