Uber will not be issued a new private hire licence, Transport for London (TfL) has said.
TfL concluded the ride-hailing app firm was not fit and proper to hold a London private hire operator licence.
It said it took the decision on the grounds of “public safety and security implications”.
Confirming it would appeal against the decision, Uber said it showed the world “far from being open, London is closed to innovative companies”.
TfL’s concerns include Uber’s approach to carrying out background checks on drivers and reporting serious criminal offences.
Uber’s current licence is due to run until 30 September.
It has 21 days to appeal against TfL’s decision and can continue to operate while any appeals are ongoing.
Some 3.5 million passengers and 40,000 drivers use the Uber app in London.
Mayor of London Sadiq Khan said: “I fully support TfL’s decision – it would be wrong if TfL continued to license Uber if there is any way that this could pose a threat to Londoners’ safety and security.”
Fred Jones, head of cities for Uber across the UK and Ireland, told the BBC Uber drivers had to pass the same safety checks as black cab and mini cab drivers in London.
There had been growing speculation that the app could be banned from London.
Opponents of the firm claim it causes gridlocked roads and does not do enough to regulate its drivers.
But one driver with Uber in London said: “I don’t think it is a fair decision. Uber offers a flexible schedule, and a weekly income.”
Uber’s general manager in London Tom Elvidge said: “By wanting to ban our app from the capital, Transport for London and the mayor have caved in to a small number of people who want to restrict consumer choice.
“If this decision stands, it will put more than 40,000 licensed drivers out of work and deprive Londoners of a convenient and affordable form of transport.
“To defend the livelihoods of all those drivers, and the consumer choice of millions of Londoners who use our app, we intend to immediately challenge this in the courts.”
He said Uber operated in more than 600 cities around the world, including more than 40 towns and cities in the UK.
Analysis: From BBC technology correspondent Rory Cellan-Jones
Throughout its short, tempestuous life, Uber has clashed with regulators around the world – and more often than not it has come out on top.
Its tactic has often been to arrive in a city, break a few rules, and then apologise when it’s rapped over the knuckles. Some regulators have backed down, others have run the company out of town.
In London, despite protests from angry taxi drivers, the company has had a relatively easy ride until now.
But a wave of bad publicity about its corporate culture, its lax attitude to checks on its drivers and its treatment of this freelance army seems to have spurred TfL into action.
Make no mistake, Uber will use every legal avenue to fight this ban. It will argue that consumers, in the shape of the millions of mainly young Londoners who rely on its service, will be seriously let down if it can no longer operate.
But the courts will have to balance that with the serious concerns about public safety raised by TfL.
On social media, a fierce debate has broken out over the decision.
An online petition launched by Uber urging Sadiq Khan to reverse the decision to strip its London licence has been signed by tens of thousands of people in the space of a few hours.
Twitter user @Gabbysalaza_ said that she was “annoyed” at the decision as Uber allowed to her to get out of “uncomfy” situations if out at night.
Labour MP Wes Streeting, chairman of the All Party Parliamentary Group on Taxis, called the decision “courageous” in a tweet.
James Le Lacheur called the decision a “victory” on Twitter.
General secretary of the Licensed Taxi Drivers’ Association Steve McNamara said it was the “right call” not to re-license Uber in London.
“This immoral company has no place on London’s street,” he said.
Across the world, Uber has been pushed out or denied access by local licensing laws.
Legislators in Darwin, in Australia’s Northern Territory, are debating whether to allow Uber to return after a raft of reforms designed to open up the ride-sharing market were announced.
Uber is currently fighting a test case in Denmark after four if its drivers were found to be in violation of the country’s laws requiring taxi meters.
David Leam, of London First which campaigns for business in the capital, said London needed to be open to new ideas, business and services.
He said: “This will be seen as a Luddite decision by millions of Londoners and international visitors who use Uber, and will also hit London’s reputation as a global tech hub.”
James Farrar, chairman of the Independent Workers’ Union of Great Britain’s United Private Hire Drivers branch, said it was a “devastating blow” for the drivers who now face losing their jobs.
“To strip Uber of its licence after five years of laissez-faire regulation is a testament to a systemic failure at TfL,” he said.