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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".
Some 3.5 million passengers and 40,000 drivers use the Uber app in London.
In a statement, Uber said: "Transport for London and the mayor have caved in to a small number of people who want to restrict consumer choice."
Uber's general manager in London Tom Elvidge said: "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.
TfL's concerns include Uber's approach to carrying out background checks on drivers and reporting serious criminal offences.
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.
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."
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.
Mayor of London Sadiq Khan said in a statement: "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.
"Any operator of private hire services in London needs to play by the rules."
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.
General secretary of the Licensed Taxi Drivers' Association Steve McNamara said it was the "right call" not to re-license Uber in London.
"We expect Uber will again embark on a spurious legal challenge against the mayor and TfL, and we will urge the court to uphold this decision," he said.
"This immoral company has no place on London's streets."
Labour MP Wes Streeting, chairman of the All Party Parliamentary Group on Taxis, said: "This is a courageous decision by the mayor and Transport for London.
"It finally draws a line in the sand to make it clear that no company, however big and powerful, will be allowed to flout our laws and regulations or jeopardise Londoners' safety without facing serious consequences."
However, 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: "This is a devastating blow for 30,000 Londoners who now face losing their job and being saddled with unmanageable vehicle-related debt.
"To strip Uber of its licence after five years of laissez-faire regulation is a testament to a systemic failure at TfL."
Uber has 21 days to appeal TfL's decision.
Timeline: Uber
