The donation by philanthropist Peter Gilgan, the CEO of Mattamy Homes, is believed to be the largest single private gift to a children's hospital in Canada.
The hospital announced the donation Wednesday, and says it will go toward the construction and operating costs of the SickKids Centre for Research and Learning.
The $400-million facility is currently under construction in downtown Toronto, and is slated to open in August 2013.
The hospital says the laboratory and learning facility will bring together 2,000 scientists, trainees and research staff under one roof.
Gilgan's company built its first home in 1978, and he and his employees have been involved previously in raising money for the hospital.
"I have been a friend of SickKids for many years and know first-hand the critical and innovative work they do every day," Gilgan said in a statement.
"I feel privileged to be able to play a significant role in advancing the research and learning in children's health that will not only benefit my grandchildren, it will benefit many children around the world."
Gilgan is a father of eight, and a grandfather of four.
Mary Jo Haddad, president and CEO of SickKids, said the hospital is extremely grateful for the support.
"This gift is a giant step forward in our mission to advance children's health, and further enables our capacity to make groundbreaking discoveries in the years to come," she said.