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TfL said the move was to ensure all passengers felt "welcome".
Staff of London Underground have been told to say "hello everyone" in an effort to become more gender-neutral, the BBC reported.
The revised phrasing will be applied to all new pre- recorded announcements made across the British capital's transport network.
Mark Evers, director of customer strategy at TfL, said: "We want everyone to feel welcome on our transport network.
"We have reviewed the language that we use in announcements and elsewhere and will make sure that it is fully inclusive, reflecting the great diversity of London."
The London Underground is a public rapid transit system serving London and some parts of the adjacent counties.
London Mayor Sadiq Khan said he was "keen" TfL speak in a "more neutral way".
He said: "TfL serves a vibrant, diverse and multicultural city, and provision of an inclusive transport service is at the heart of TfL's purpose.
TfL said it had briefed staff on use of the new language "though from time-to-time, well-meaning staff may still use the term 'ladies and gentlemen"."
"If this happens frequently, we will issue reminders to staff," it added.
LGBT campaign group Stonewall welcomed the decision.
Stonewall said: "Language is extremely important to the lesbian, gay, bi and trans community, and the way we use it can help ensure all people feel included.
"We welcome gender neutral announcements to be rolled out across TfL as it will ensure that everyone - no matter who they identify as - feels accounted for," it added.
(This story has not been edited by Business Standard staff and is auto-generated from a syndicated feed.)