The regulator said the taxi app was not “fit and proper” as a licence holder, despite having made a number of positive changes to its operations.
Uber originally lost its licence in 2017 due to safety concerns, but was granted a 15-month extension.
The firm says it will appeal against the decision and can continue to operate during that process. It had received an additional two-month extension in September which expired on Sunday.
Uber has faced resistance from regulators and traditional taxi services in a range of countries after being dogged by controversy for a number of years.
Helen Chapman, Director of Licensing, Regulation and Charging at TfL, said:
“As the regulator of private hire services in London we are required to make a decision today on whether Uber is fit and proper to hold a licence. Safety is our absolute top priority.
“While we recognise Uber has made improvements, it is unacceptable that Uber has allowed passengers to get into minicabs with drivers who are potentially unlicensed and uninsured.”