It happened just before midnight, during a screening of the comedy We're The Millers.
Police say 150 people were evacuated from the theatre. Seven people were treated at the scene by fire crews, who used decontamination gear.
Laura Weiss was in the theatre and said she was shaken up by the incident.
"Some people just left the theatre and were kind of laughing it off and they just kind of walked out," said Weiss who was inside the theatre. "I was freaked out so I was staying to see, kind of, what the word was on what happened," she said.
Weiss said people tried to get out the emergency exits, but they were blocked because of construction in another part of theatre.
Police said video surveillance from inside the theatre links a woman and two men to the bear spray attack.
"We do have surveillance video that we are looking at and we are trying to determine a better description, a more specific description of the three people we believe are suspects here," Saskatoon police spokesperson Alyson Edwards said.
It's legal to purchase bear spray, but police say they're investigating this as an assault.
"It's a concern to us," Edwards said. "It does injure people. This is an assault investigation. And we've never seen it before where it's been deployed inside a theatre where people are in a confined space."
Cineplex Entertainment, the company that owns Galaxy Cinemas, released a statement regarding the incident.
"Theatre employees followed protocol, contacting police immediately after the incident occurred," said Mike Langdon, director of communications for Cineplex Entertainment, in the statement. "We have been working closely with the authorities to identify the individual(s) involved."
Investigators are asking anyone with information about the suspects to contact them.