Pre-recorded messages will also be changed to reflect the new language.
The shift in wording comes after months of campaigning by LGBTQ activists, who urged London Mayor Sadiq Khan to scrap the binary "ladies and gentlemen" greeting.
Khan asked for a review of TfL training and procedures in December after a trans woman said she was ridiculed by a transport worker who said she "didn't sound like a Miss" during a call to the agency's helpline.
TfL's head of customer strategy Mark Evers says the new language aims to make "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," he told the Evening Standard.