Filming wasn't taking place on the 007 stage when the blaze broke out at the Pinewood Studios, west of London.
The fire brigade said gas canisters may have exploded inside the building, causing the roof to partly cave in.
No one was reported injured in the fire, which took fire fighters 90 minutes to bring under control.
Sunday's blaze came 22 years after Pinewood Studios' 007 stage was burned to the ground during filming.
"We do not know the extent of the damage to the 007 stage, although it is believed to be significant", Pinewood said in a statement.
Eight fire engines were called to battle the blaze which could be seen up to 16 kilometres away.
Television pictures showed large plumes of black smoke billowing from the collapsed roof, which was made of corrugated steel.
Eyewitness Jen McVean said there was no doubt the building had been badly damaged.
"There was a very big plume of smoke, and we went into the studios to check, and we could see the Bond stage was on fire," said McVean, who owns a special effects company at Pinewood.
The vast stage had been transformed into a replica of the Italian city of Venice, where part of the film is set.
'Casino Royale', due for release in October, will be the first featuring new James Bond actor Daniel Craig.
Craig was chosen last year to replace Pierce Brosnan as British super-spy Bond.
The 007 stage was first created in the late 1960s for the filming of "The Spy Who Loved Me" and was created when the script called for filming of two submarines inside an oil tanker.
The stage was built complete with an enormous water tank.
Other Bond scenes filmed around the studio include a car chase in Goldfinger's factory, and Spectre Island in "From Russia With Love".
Bond films at the studios date back to 1962 when "Dr. No" was filmed.
Pinewood studios, which are set within a former country estate, act as the base for the James Bond films although in recent years more filming has been done on location to reduce costs.