Victorian members of the Australian Education Union voted to turn down the state government's updated offer of a 28.3 per cent pay rise over the next four years.
The union on Tuesday revealed almost 49 per cent of members voted to accept the deal and more than 51 per cent of members rejected it.
The union's Victorian president Justin Mullaly said the outcome sent an undeniable message to the government that change was needed.
"Members have been telling the Victorian Labor government for years that the under-resourcing of public schools in Victoria is making their work untenable," he said in a statement.
"When just three in 10 public school teachers plan to stay working in public schools in Victoria, it is obvious there is a problem."
Union educators have been locked in a pay dispute with the government for months, with graduate teachers - the worst paid in the country - earning $13,000 less than those in the Northern Territory, who are the best paid.
Members had been pushing for a 35 per cent pay rise over four years after rejecting an initial offer of an 18.5 per cent increase over three years.
More than 35,000 Victorian teachers, principals and education support workers walked off the job in March, the first strike of its kind in 13 years.
In June, teachers voted down a revised state government offer on pay and conditions, despite their union reaching an in-principle agreement with the government.
A fresh statewide strike was announced by teachers for Thursday, prompting the government to table another an eleventh-hour pay offer.
Voting on the second revised proposal closed on Tuesday afternoon, with the result determining whether teachers would strike.
More than 50,000 members took part in the vote.
Members will push forward with the strike, gathering in Melbourne's CBD.
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