Neither the name of the man nor the identity of the 12-year-old has been released, but police say they are related.
Homicide detectives did not provide many details about the incident Wednesday, but said a "family situation" led to the stabbing.
Police responded to the scene at the Cambridge Suites Hotel, near the intersection of Richmond Street East and Victoria Street, just before 6 a.m. ET.
They found the boy in life-threatening condition. He was taken to hospital, where he later died.
The boy had been staying at the hotel with his family, police said.
The man was set to appear in Toronto's College Park courthouse at 10 a.m. ET Thursday.