On a day of sentimental successes and broken records, it was the first time a reigning monarch's horse had claimed victory in the race's 207-year history.
Estimate, a four-year-old filly trained by Michael Stoute, held off a challenge by Simenon in the final furlong to win by a neck and initiate a rapturous reception for the Queen in the winner's enclosure.
The Queen was expected to present the winner's trophy, but instead received it from her second son, the Duke of York.
Estimate, a 7-2 favourite, won at Royal Ascot last year when landing the Queen's Vase.
The Queen's grandson Peter Phillips told Channel 4: "It's amazing! This is her passion and her life.
"She's here every year and she strives to have winners -- to win the big one at Royal Ascot means so much to her."
Jockey Ryan Moore told reporters: "To win the Gold Cup in the Queen's colours is exceptional."
Stoute said: "I really felt it was a tough task -- I wasn't confident at all with her taking on the boys.
"But it's a great thrill. Estimate is a pain in the backside at times, but it's a great honour and today is very special."
Earlier, in emotional scenes after the Ribblesdale Stakes, there was universal acclaim for the victorious Riposte, trained by Jane Cecil, widow of master trainer Henry Cecil who died on June 11, having sent out a record 75 Ascot winners.
"First of all, that was for Henry, the Prince (Saudi Arabian owner Khalid Abdullah) and all the staff at Warren Place - for team Cecil. I don't really have the words to say what I am feeling right now," Cecil said.
"Henry was just adored by so many people. Even people who had never met him just loved him."
In another thrilling race, American sprinter No Nay Never broke the five-furlong track record by a two-year-old in the Norfolk Stakes.
The Wesley Ward-trained son of U.S. sire Scat Daddy recorded a time of 58.80, improving on the previous best of 59.17 despite a slow start.
"He's like a bull," said Ward. "I knew he would push his way through and, while it was a bit of a concern, we'll take him home and work on that. He'll be better at the gate next time."
10 No Nay Never 4/1
2 Coach House 9/4 F
14 Wind Fire 16/1
9 Riposte 9/2
8 Just Pretending 9/2
3 Elik 9/2
18 Estimate 7/2 F
9 Simenon 5/1
17 Top Trip 7/1
7 Roca Tumu 20/1
21 Tarikhi 33/1
11 Queensberry Rules 16/1
13 Wentworth 7/2 F
8 Remote 9/4 F
10 Shikarpour 7/1
11 Van Der Neer 14/1
13 Elidor 20/1
8 Space Ship 16/1
10 Bold Sniper 7/2 F
1 Cap O'rushes 20/1
- Sports & Recreation
- Royal Ascot
- Michael Stoute
- Henry Cecil