Anyone wanting to become a driver for the billion-dollar startup, or any other minicab firm in the capital, will have to provide evidence of their English ability.
The new rules were introduced over the weekend and are part of wide ranging reform across the private hire industry following a high profile consultation.
However, the rules are facing a backlash from Uber which successfully won a judicial review of some.
It is awaiting a hearing on several matters, including claims that the English language requirements are discriminatory.
However, the final detail of the new rule shows it will apply to anyone requesting a license and will not be based on nationality - something Uber had argued would disproportionately impact certain nationalities and on black and ethnic minorities.
TfL said in its latest notice that it no longer intended to proceed with "an exemption that would apply to those from a majority English speaking country".
Now, any person wanting to become a driver, whether they are from the UK or elsewhere, as well as current drivers renewing their license, will have to have documents proving they have the correct level of English skills in speaking, listening, reading and writing.
Drivers must have B1 level English, or the equivalent of a GCSE in the subject.
Anyone reapplying for a license will have until the end of March next year to provide evidence of their language skills as a transition phase.
“It’s disappointing that, to try and dig themselves out of a legal hole, TfL is now insisting every private hire driver in London must have essay writing skills," said an Uber spokesperson.
“We’ve always supported spoken English skills, but passing a written English exam has nothing to do with communicating with passengers or getting them safely from A to B. Thousands of drivers who’ve spent years providing a great service to Londoners will now have to fork out £200 and pass a writing exam, try to find an old GCSE certificate or lose their licence and their livelihood."
The firm believes the rules are still unnecessary and called for them to be scrapped.