A 13-year-old Castlemaine girl was airlifted to hospital following an accident near the intersection of Duke and Urquhart Streets just outside the Woolworths Castlemaine precinct on Monday evening.
It is believed a black Nissan X trail and an e-bike collided on Urquhart Street about 5.30pm.
Police and emergency services responded to the collision.
The driver of the vehicle stopped immediately at the scene.
Officers were told two people were riding the e-bike at the time.
A 12-year-old boy from Campbells Creek was hospitalised with non-life-threatening injuries. A 13-year-old girl from Castlemaine was transported to the nearby Western Reserve where she was airlifted to hospital with life-threatening injuries.
The circumstances are yet to be established and an investigation into the collision is ongoing.
Investigators from the Major Collision Investigation Unit re-attended the scene of today to conduct further investigation.
Anyone who witnessed the incident, has CCTV or dashcam vision or information is urged to contact Crime Stoppers on 1800 333 000 or visit www.crimestoppersvic.com.au