Two teenage girls and two adults were seriously injured in the accident on the Western Highway at Bacchus Marsh in the early hours of Wednesday morning.
The school bus was carrying four adults and 27 students in years nine to 11 from Ballarat's Loreto College to the airport for a trip.
VicRoads were clearing a crash from earlier in the night when the truck collided with the bus, police said.
"Quite miraculously, they've self-evacuated and other people, I believe truck drivers and that, stopped to assist them," Detective Inspector Roger Schranz told reporters on Wednesday.
"I would have assumed someone would have passed away out of this entire tragedy. So they're all very fortunate people."
Two teenage girls were flown to the Royal Children's Hospital in a serious but stable condition.
A woman in her 40s and a man in his 50s were taken to the Royal Melbourne in a serious but stable condition.
18 others were taken to various hospitals in a stable condition.
Police will investigate whether drugs, alcohol or speed were factors in the crash.
Melbourne-bound highway lanes are expected to be closed for the rest of the day as investigators analyse the scene.
The students' parents were asked to stay away from the crash and contact Ballarat police station.
Nearby resident Ange Greenland woke to the sound of the air ambulance arriving.
She told AAP the crash site was "pretty nasty" and it was distressing that the young girls had to walk up the embankment after the crash.
"They walked up the off ramp to a waiting bus half a kilometre away," she said.
"I really was very angry because I thought if that was mine, if it was my child ... I could not believe how they were treated".
In a statement the school thanked emergency services for coordinating care and providing triage at the scene.
"Our caring Loreto community has deep concern for the injured and their families and we ask for their privacy to be respected," it said.
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