The 21 girls, aged seven to 12, will have separate duties to the boys but will combine with them for important occasions like Christmas services.
Conductor Nia Llewelyn Jones, said: "They're not that different to the boys - cheeky, sassy, but they're a great bunch of girls."
The cathedral's choir was founded in 1541 by King Henry VIII.
Since 1999 girls have been able to sing, with the boys, in the Cathedral Youth Choir for 12 to 18 year olds.
Then in 2014, the Cathedral Junior Choir was set up for both boys and girls - aged six to 12.
Canon Celia Thomson said: "I hope that lots of people will come to hear them and experience the worship with them singing."
The Girl Choristers of Gloucester Cathedral Choir will hold their inaugural Evensong at 17:30 GMT.
An all-girl choir is to sing for the first time in Gloucester Cathedral Choir's 475-year history.