Sarah Jessica Parker, Idina Menzel and Carole King are among those involved in the initiative, inspired by the 1985 We are the World benefit for Africa.
"Everyone wanted to do something as quickly as possible," said instigator Seth Rudetsky, a musical director.
The digital download, available from Monday, will cost $1.99 (Â£1.40).
Matthew Broderick, Gloria Estefan, Whoopi Goldberg and Rosie O'Donnell are among a company of about 60 who will be "raising their voices in song" in New York, Los Angeles and Miami.
Lin-Manuel Miranda, who wrote and starred in Tony-winning musical Hamilton is also taking part, along with  other cast members to help re-record the Burt Bacharach-Hal David standard.
"We started making calls and suddenly we had tons of theatre actors along with an orchestra and the services of a fully-staffed recording studio, all willing to donate their time and talent," Rudetsky continued.
"Our community of artists has banded together as we always do, to show we can end this cycle of violence and intolerance."
Forty-nine people were killed and many more were injured on Sunday morning when gunman Omar Mateen opened fire at the Pulse nightclub in Orlando, Florida.
All proceeds from the sale of the song, released by Broadway Records, will benefit the LGBT Center of Central Florida.
Broadway stars past and present are to record a version of What the World Needs Now Is Love, to support victims of the Orlando nightclub shooting.