Performers from the comic opera L'elisir d'amore gathered outside the Bow Street venue in full costume at 20:30 BST, alongside members of the audience.
The theatre said the interruption was a false alarm and the show had restarted.
People took to Twitter after being caught up in the real-life drama.
Grace Mallon tweeted: "Exciting times at Elisir d'Amore @RoyalOperaHouse - first time I've been evacuated for a fire alarm in the middle of a performance!"
Matt Innes‏ tweeted: "Delightful Donizetti interrupted for a fire alarm, but about to restart to a cheer. Blitz spirit right here."
Michael Amon‏ added: "#royaloperahouse big says that wasn't a drill and thanks everyone for a "brilliant" evacuation. Ready to start in a couple minutes."
Cast members and theatre-goers were evacuated from the Royal Opera House in London after a fire alarm went off mid-performance.