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UK Anti-Doping said Khan, 36, returned a positive result for the anabolic agent ostarine after losing to Brook in Manchester on February 19 2022.
Khan, who announced his retirement in May last year, accepted a violation of anti-doping rules but said he had not intentionally ingested the substance, an argument accepted by an independent panel following a hearing in January.
A UKAD statement said: "Mr Khan's two-year ban is deemed to have commenced on April 6 2022 (the date his provisional suspension was imposed) and will expire on April 5 2024."
When asked about the verdict by Sky News, Khan was defiant, replying: "I have never cheated.
"I am a retired fighter. You can see by my performance against Kell Brook [that it] wasn't the best. I lost the fight. If I went in there and knocked Kell Brook out it is different. I have never cheated in my life, I am the one who wanted the testing done.
"The amount that was in my system could have come from shaking peoples' hands. I don't know what the drug was in my system. I will give my views, but, like I say, I have never cheated in my life. I would never cheat. I am a retired fighter, and I have got this two-year ban now, which is quite strange. I have already retired anyway. No comeback plans at all."
Khan was stopped in the sixth round of his grudge fight with bitter rival Brook 14 months ago in what proved to be his final professional bout.
An independent tribunal accepted Khan's argument and ruled out "deliberate or reckless conduct" in a written decision dated February 21 2022, and imposed a two-year ban, rather than a more severe four-year suspension, from all sport due to "strict liability".
"Strict liability means athletes are ultimately responsible for what they ingest and for the presence of any prohibited substances in a sample," UKAD chief executive Jane Rumble said.
Ostarine is a selective androgen receptor modulator that can help muscle growth and, according to UKAD, is designed to have similar effects to testosterone
Khan unified world titles at super-lightweight and faced pound-for-pound stars Saul 'Canelo' Alvarez and Terence Crawford during a 40-fight career, featuring 34 wins and six defeats.
The Bolton fighter had launched his pro career in 2005 after winning an Olympic silver medal at the 2004 Athens Games.
Khan became a world champion in July 2009, defeating Andriy Kotelnik on points to secure the WBA super-lightweight title, and he then stopped Zab Judah to add the IBF belt two years later.
A hotly-disputed points loss to Lamont Peterson ended Khan's world title reign, and he was halted by Danny Garcia while attempting to win the WBC and WBA belts in his next fight.
Khan made an audacious move up to middleweight to challenge Canelo for the WBC belt, suffering a sixth-round knockout loss in May 2016 and was stopped by Crawford in the sixth round of their WBO title fight in April 2019.
He eventually shared the ring with Brook in February 2022, embracing his rival in the ring after their feud was finally ended.
BOXXER 'condemns such unsporting behaviour'
A spokesperson for BOXXER, who promoted the Khan vs Brook fight, said: "BOXXER is disappointed to only learn via social media this morning that Amir Khan has been suspended from all sports following UK Anti-Doping (UKAD) rule violations.
"BOXXER is vehemently against any use of any illegal or performance enhancing substances taken by athletes and condemns such unsporting behaviour. It goes against the very core of our company's values. We will await further advice from all the relevant authorities."