Friday's decision to ban the ride-sharing service was "affecting the real lives of a huge number of honest and hard-working drivers" and would "show the world that London is far from being open and is closed to innovative companies," the petition said.
Transport for London said the conduct of Uber, which has around 40,000 drivers and 3.5 million customers in the British capital, had raised safety concerns.
"TfL has concluded that Uber London Limited is not fit and proper to hold a private hire operator licence," it said in a statement.
It said Uber's "approach and conduct demonstrate a lack of corporate responsibility in relation to a number of issues which have potential public safety and security implications", citing concerns over background checks of drivers.
The petition, on change.org, said that Uber provided a "safe, reliable and affordable ride", and thar its users would be "astounded" by the ban.
"By wanting to ban our app from the capital, Transport for London and their chairman the Mayor have given in to a small number of people who want to restrict consumer choice," it added.
The licence expires on September 30 but Uber has 21 days to appeal the decision, and has said it plans a challenge.
Labour Party leader Jeremy Corbyn on Saturday backed his London mayor Sadiq Khan, telling Sky News that authorities had done the "right thing".
"Obviously people need to be able to travel, obviously they want to be able to access cabs," he said. "Those cabs must be safe, must be regulated and must be available for all."
