The midnight tally stood at $15,820,640.78 eclipsing last year's record of $15.1 million.
The appeal has received $230 million in donations from the Victorian community since it began in 1931.
Retiring Executive Director Christine Unsworth said the response had been overwhelming.
"We are over the moon. This is a amazing amount of money for the kids," she said in a statement on Saturday.
Ms Unsworth said she felt privileged to be part of the appeal over more than 16 years and thanked volunteers, donors and corporate sponsors.
"Victorians and Australians are rightly very proud of the Royal Children's Hospital and the amazing work it does and this is reflected in their generosity."
Volunteers with collecting tins hit the streets in a bid to break the record, while sport and television stars, as well as the Easter bunny, spent the day with children at the hospital who are unable to be home for Easter.
Ms Unsworth said rural and regional collectors raised $3.6 million with an online auction raising a record $147,950.
Firefighters from the CFA and MFB took part in a 24-hour relay in the CBD to help the hospital reach its goal.
Ms Unsworth said the thousands of CFA tin rattlers around the state raised $1.25 million.
Money raised from the appeal will fund state-of-the art medical equipment, ground-breaking research and scholarship opportunities.
One item high on the shopping list is a MR/PET machine, costing $8 million.
This machine combines MRI and PET scans and would be the first in a pediatric hospital anywhere in the world.
Hundreds of Victorian schools also raised money for neo-natal cots that cost $29,000 each.
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