The bus was slowing down to avoid an earlier crash when it was hit, leading to two teenage girls and two adults being seriously injured.
The school bus was on the Western Highway at Bacchus Marsh in the early hours of Wednesday morning carrying four adults and 27 students in years nine to 11 from Ballarat's Loreto College to the airport for a trip.
"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 Hospital in a serious but stable condition.
Eighteen others were taken to various hospitals in a stable condition.
Ballarat Base Hospital took on 16 patients from the crash, with operator Grampians Health declaring a "code brown" to divert resources to its emergency department.
By 2pm on Wednesday, it dropped the code brown declaration, and an hour later, 13 patients had been discharged. The remaining three were admitted to the hospital and in a stable condition.
"We were very lucky here with seatbelts," Grampians Health chief executive Dale Fraser said.
"Our site sees far too many people every single day who run the risk of serious permanent harm or even death through road traffic accidents. I would love not to see them in our hospital."
Police will investigate whether drugs, alcohol or speed were factors in the crash.
Melbourne-bound highway lanes were expected to be closed for the rest of the day as investigators analysed the scene.
The students' parents were asked to stay away from the crash and contact Ballarat police station.
The coach operator said in a statement their driver managed to get everyone out of the bus before being taken to Austin Hospital for shock.
The owner of Little's Coaches also personally took 11 students and some parents to Ballarat Base Hospital in a separate bus following the crash.
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.
The school would remain open on Thursday despite the public holiday to allow students and families to come together and receive counselling, principal Michelle Brodrick said.
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