Premier Kathleen Wynne announced the funding, which involves the province covering 100% of the capital costs of building the LRT.
Light rail vehicles on tracks separated from regular traffic will link McMaster University through downtown Hamilton to Queenston Circle.
Procurement will start in 2017, with construction getting under way in 2019.
In the 2015 budget, the government committed to investing in rapid transit in Hamilton as part of the Moving Ontario Forward plan. Through this plan, Ontario will invest CA$31.5bn over 10 years in public transit, transportation and other priority infrastructure projects across the province.
"Our investment in the Hamilton LRT will help support the exciting revitalisation under way in Hamilton,” said Wynne. “We are making record investments across Ontario in infrastructure projects like this to strengthen our economy and quality of life - and lay the groundwork for future prosperity."