Both Charles and Camilla wiped tears away in the Royal Box as Tom Marquand got their horse past Valiant King to take the honours.
They descended from the box to the winner's enclosure, the king with a broad smile on his face as he shook hands with Marquand and trainer William Haggas.
The royal couple then accepted the trophy from the Duke of Kent with Princess Anne and her daughter Zara Tindall looking on.
"I am thinking of how proud the Queen would have been for Charles and Camilla to have a winner and keep the dream alive," said Zara.
"It is a new excitement for them. Like all those owners who have a horse here they are living that dream and hope and fulfilling it is incredible."
For Haggas - who had lunch at Windsor Castle with Charles and Camilla on Wednesday - it was a wonderful experience.
"Absolutely fantastic, it is a great honour and I am thrilled they were here to see it," said Haggas.
Due to protocol, those conversing with the monarch are forbidden from relaying what is said but 62-year-old Haggas said the importance of the victory was immense.
"They have been looking forward to Ascot for a long time and wanted to have as many runners as possible and they will be absolutely delighted," he said.
"It is very important for them to have a winner and that they enjoy it."
Marquand is normally pretty restrained in celebrating but he let himself go after this landmark victory, punching the air and grinning and chatting to spectators as he came back to the enclosure.
"This is one of proudest moments of being in the saddle," he said. "I grew up watching horses like this win for Her Majesty the Queen.
"I am in a dream world... Royal Ascot is the pinnacle and riding a royal winner is so special. It is an emotional day."
Charles and Camilla still have some way to go to overhaul Queen Elizabeth II, who had 24 Royal Ascot winners, including Estimate in the week's most historic race, the Ascot Gold Cup, in 2013.
