The 29-year-old, who won gold in Beijing in 2008, revealed her decision at a press conference in London and blasted the dopers who have blighted the sport.
"I am very happy with my career. I have many, many happy memories over what has been my life's work since I was 12," said Cooke.
"I have won every race and more that I dreamed I could win. I am so very fortunate to have been able to have won clean. I have been robbed by drug cheats, but I am fortunate, I am here before you with more in my basket that the 12 year old dreamed of.
"But for many genuine people out there who do ride clean, people with morals, many of these people have had to leave the sport with nothing after a lifetime of hard work.
"When Lance (Armstrong) cries on Oprah later this week and she passes him a tissue, spare a thought for all of those genuine people who walked away with no reward.
"Tyler Hamilton will make more money from a book describing how he cheated than I will make in all my years of honest labour."
The Welsh star won the British road race on 10 consecutive occasions, claimed gold at the 2002 Commonwealth Games, triumphed in La Grande Boucle Féminine – the women’s Tour de France - in 2006 and 2007 and also won the World Championship road race in 2008.
But Cooke, who was awarded an MBE in 2009, has not won a UCI race since April.
"I hope I will look on in 10 years' time and see a vibrant and healthy women's road scene. The key to that will be that the female athletes are treated with respect," she concluded.