“The plane has taken off, everyone is on board,” said the Russian embassy in Washington, quoted by the state-owned Ria Novosti agency.
Relatives of the diplomats are also onboard, making 96 in all.
The expulsions were part of a package of sanctions ordered by President Barack Obama on Thursday in the final weeks of his administration.
US intelligence says the Kremlin ordered a hack-and-release of Democratic Party and Hillary Clinton campaign staff emails in a bid to put Donald Trump in the Oval Office.
Thirty-five Russians, described as intelligence operatives based at the Russian embassy in Washington and the consulate in San Francisco, were given 72 hours to leave the country.
Obama also ordered the closure of two Russian compounds in New York and Maryland that the United States says are used “for intelligence-related purposes.”
Economic sanctions were also announced against Russia’s FSB and GRU intelligence agencies. Four GRU officers including agency chief Igor Korobov also face sanctions.
Moscow has repeatedly denied the allegations.
President Vladimir Putin has ruled out sending home US diplomats in retaliation — a move interpreted as a sign he is looking to Trump to rebuild US-Russian ties after the US inauguration on January 20.