It comes after months of debate over passenger safety and vetting of drivers.
Withdrawing the licence, TfL described Uber as not being “fit and proper” to offer its services to Londoners.
Mayor Sadiq Khan said the decision had been made to protect customer safety, adding that all companies must “play by the rules”.
The firm criticised the move, saying that “far from being open, London is closed to innovative companies”.
Uber’s London licence will expire on September 30.
The company can continue to operate beyond the expiry date, however, if it decides to appeal the decision.
In deciding whether to renew the licence, TfL considered the mincab app’s approach to reporting serious criminal offences and how it carried out background checks on its drivers.
Mr Khan said: “I want London to be at the forefront of innovation and new technology and to be a natural home for exciting new companies that help Londoners by providing a better and more affordable service.
“However, all companies in London must play by the rules and adhere to the high standards we expect – particularly when it comes to the safety of customers. Providing an innovative service must not be at the expense of customer safety and security.
“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.”
Uber’s general manager in London confirmed it intended to appeal the decision.
Tom Elvidge said: “3.5 million Londoners who use our app, and more than 40,000 licensed drivers who rely on Uber to make a living, will be astounded by this decision.
“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.”
Mr Elvidge insisted the company abided by the rules and carried out “enhanced” background checks on its drivers.
The decision is likely to be welcomed by Uber’s growing critics, who argue the company has exacerbated London’s traffic congestion and done little to address incidents of violence involving its drivers.
Labour MP Wes Streeting, chairman of the All Party Parliamentary Group, described the decision as “courageous”.
“Uber has not shown itself to be a fit and proper operator,” he said.
“It stands accused by the police of failing to properly handle serious allegations of rape and sexual assault of passengers.
“It had to be dragged through the courts to recognise its responsibility to provide even the most basic rights and protections to Uber drivers.
“Its business model is based on saturating London’s taxi and private hire market to drive its competition off the road.”
Meanwhile, Steve McNamara, general secretary of the Licensed Taxi Drivers’ Association said TfL had made the “right call”.
“Since it first came onto our streets Uber has broken the law, exploited its drivers and refused to take responsibility for the safety of passengers,” he said.
“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.”
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.
Uber enables users to book cars using their smartphones, and is available in cities across the UK.
Some 3.5 million passengers and 40,000 drivers use the Uber app in London.