The charity was one of over 3,000 nominations for the prize, and made it through to the 24 finalists. It won the public vote, in which over 130,000 votes were cast.
The charity was set up by Katherine and Jamie Staples following the death of their son Harley in November 2009. He died from a rare and aggressive form of leukaemia at the age of eight.
They are aiming to raise £500,000 to build Harley's House, a holiday home to give young cancer sufferers time away from hospitals and treatment. They have already raised £250,000, so winning the house will get them to their target.
Jamie Staples said: "We are stunned and speechless. We have achieved our dream and created a true legacy for Harley. Thanks to everyone who has supported us. It's blown us completely away and is a huge achievement".
All the 23 other finalists received a donation of £6,000 each from Persimmon Homes. The finalists included Yorkshire's Forget Me Not Children's Hospice, Birmingham's St Basil's, Scotland's Zac Scott Braveheart Foundation and The Kent Multiple Sclerosis Therapy Centre.
How did they win the vote?
The Harley Staples Cancer Trust's campaign included a video, Vote for The Harley Staples Cancer Trust, which was the most watched Persimmon charity film on YouTube with nearly 2,000 views.
They were helped by band Kasabian who made an announcement at the Reading festival to urge people to vote.
Katherine and Jamie also Staples also made a half-time appeal at the King Power Stadium in front of thousands of Leicester City and Blackpool fans.
Further support for their campaign came from the Leicester Mercury newspaper, cricketer Matthew Hoggard, Leicester City football manager Nigel Pearson and football legend Steve Walsh.