Detectives from Victoria Police's major collision unit are investigating the crash at Diamond Creek, in Melbourne's outer northeast.
Emergency services were called to Chute Street about 10pm on Monday after reports of a collision between a 4WD and an electric motorcycle.
The rider of the bike, believed to be a teenage boy, was treated at the scene but could not be revived.
The vehicle's driver is assisting police as officers investigate the circumstances surrounding the collision.
The boy's death is the latest in a spate of fatal e-bike crashes across the nation, which have triggered calls for a safety crackdown on the popular means of transport.
In Sydney, an e-bike rider died a week earlier after being hit by a garbage truck.
He was not wearing a helmet.
In Brisbane, a 15-year-old boy was charged over an e-bike crash that killed an eight-year-old boy in October.
State politicians have been pleading with the federal government to clamp down on the import and sale of e-bikes able to reach dangerously high speeds amid a spate of crashes.
In a Monash University study published in August, researchers found many e-bike delivery riders in Melbourne were breaking the law, including some reaching speeds of up to 54 km/h.
One in five exceeded Victoria's e-bike speed limit of 25 km/h, the research, commissioned by the Victorian Automotive Chamber of Commerce (VACC), found.
Most of the 27,000 e-bikes observed were not using pedals, indicating they were throttle-powered or illegally modified, according to the research.
It urged authorities to look at the legal definition of e-bikes, enforce existing road rules for non-compliant devices, and close import loopholes.
WeRide, a group representing bicycle and e-bike companies, said leadership from the federal government was needed to ensure e-bikes entering Australia met local standards.
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