Hooper, who was attempting to make the Australian Sevens team for the Olympic Games, announced on social media that his attempt had ended when he missed the side.
"What a ride! My Olympic journey has come to an end, and with it, my Australian rugby career."
A veteran of 125 Tests and, at 23, the youngest player to captain the side, he reached 100 Tests for Australia and led the side 69 times, a record. He played his last Test last year, against South Africa, before switching to Sevens.
Hooper made his Test debut in 2012 against Scotland.
After starting in Super Rugby with the Brumbies, he switched to the Waratahs in 2013 and shared the Waratahs' Super Rugby title win in 2014. He was four times a winner of the John Eales medal.
Rugby Australia chief executive Phil Waugh said Hooper was an 'incredible player and ambassador' for the game.
"He leaves a legacy that will remember him as one of the greatest to wear the Wallaby gold, and a favourite among Australian supporters."
During the weekend, Beale suffered an Achilles tendon injury while playing for his Sydney club, Randwick.
The extent of his injury will be seen after scans on Monday, but it is a blow for the 95-Test veteran who hoped to return to Test rugby against Wales on Saturday.
Fullback contender Andrew Kellaway said, "It's horrible to see, but for KB, I grow up watching him and eventually playing with someone who I now consider a close mate; it sucks to see after he was right back on the brink of where he used to be.
"We were all excited to have him back and, touch wood it's not too serious."