The ambulance was in high speed and hit a truck ahead of it when the latter's driver suddenly applied brakes, Fatehpur SHO Sadar Rampratap said.
A 23-year-old youth Chelu Singh, who was already injured and was being taken to SMS hospital from Bikaner for treatment died in the incident, he said.
Chelu Singh's mother Sipu Devi and cousin Bhairon Singh also died, the SHO said.
The bodies were handed over to family members after the post-mortem.