Transport for London (TfL) has designated Uber "not fit and proper" to offer a service in London, refusing the company a new license.
Uber had originally lost its license in 2017, but was granted two extensions, the most recent of which expired on Sunday.
The decision follows concerns over the identification of drivers.
TfL found that more than 14,000 trips have been made by different drivers than those shown on the app.
The regulator also found that dismissed or suspended drivers had been able to create other Uber accounts and carry passengers.
Helen Chapman, director of licensing, regulation and charging at TfL, said: "While we recognize Uber has made improvements, it is unacceptable that Uber has allowed passengers to get into minicabs with drivers who are potentially unlicensed and uninsured."
The app-based company has proved popular in London (Credit: VCG)
In a statement, the mayor of London, Sadiq Khan, said: "I support the decision by TfL's licensing official today in relation to Uber and completely understand why the decision was taken.
"Keeping Londoners safe is my absolute number-one priority and TfL have identified a pattern of failure by Uber that has directly put passengers' safety at risk."
The private hire company sent an email to customers saying it disagreed with the decision and would appeal.
Uber CEO Dara Khosrowshahi responded to TfL's decision in a tweet, saying: "We understand we're held to a high bar, as we should be. But this TfL decision is just wrong.
"Over the last two years, we have fundamentally changed how we operate in London. We have come very far – and we will keep going, for the millions of drivers and riders who rely on us."
Uber employs 45,000 drivers in London, which is one of its top five markets globally. According to the private hire company, 26 percent of its sales comes from five cities, one of them being London.