Brighton and Hove City Council said the taxi app is not "fit and proper" as it cited data breach concerns and the use of drivers from outside the area. Uber has 21 days to lodge the appeal.
Brighton is the third United Kingdom city to revoke Uber's license to operate after London in September and York in December a year ago.
Brighton & Hove council was also concerned that Uber had demonstrated a "lack of commitment" in using only licensed drivers in the city.
The decision, made unanimously at a hearing on 23 April, echoes that of Transport for London, which turned down Uber's application to renew its licence in the capital last September.
Uber drivers that are licensed with Brighton & Hove can continue to operate in the city while Uber appeals the decision.
When UBL first applied to operate in the city in 2015, it gave a "firm commitment" to adhere to the council's Blue Book taxi guidelines, and to only use Brighton and Hove licensed drivers, she said, adding the panel did not feel the spirit of the commitment had been honoured. That's partly because Brighton has stringent hurdles for private hire operators to clear before they are granted a license. However, such "cross-border" working has become a growing issue with ride-hailing services such as Uber.
Uber has lost its license in a third British city.
Brighton said out-of-town drivers posed a risk to public safety, with regulations elsewhere failing to meet its own standards, including safety aspects such as CCTV in cabs. The firm has complied with various initiatives in London to improve safety, and its appeal is due to be heard in the high court in June.
It emerged last November that Uber, which has 75 million users across the world, had concealed a hack that affected 57 million customers and drivers in 2016.