The crash happened around 4:26 pm in the area of Valentine and Shields.
Minutes earlier, the Fresno County Sheriff's Office says a deputy tried to stop the motorcycle at McKinley and Feland.
Officials say the man on the motorcycle refused to stop, leading to a short chase at legal speeds.
Investigators say the motorcyclist ended up running a stop sign and crashing into the side of a vehicle.
The motorcyclist and the woman on the bike with him were both taken to the hospital, where he later died.
The woman remains at the hospital in critical condition.
The driver of the vehicle was also taken to the hospital but is expected to be okay.
The Fresno Police Department has taken over the investigation because the crash happened in the city.
Stay with Action News for the latest updates on this developing story.