The star allrounder lasted less than 15 minutes at the SCG on Wednesday  morning before pulling up sore midway through bowling an over.
A disappointed Stokes, who has a history of groin injuries, appeared hobbled as he left the pitch to applause from the crowd
As of 10.45am, Stokes had not officially been ruled out of the game and was being assessed for an injury to his adductor muscles, located in the groin.
"Ben Stokes is currently being assessed for a right adductor complaint. We will provide an update when more information is available," an England representative said in a statement provided to AAP.
His substitution from the game came after teammate Jacob Bethell conceded the seven English players who had featured in all five Tests this summer were by now mentally and physically fatigued.
England have endured a tough summer, with hopes of retaining the Ashes urn slipping away inside three Tests totalling 11 days of cricket.
The injury will no doubt raise questions as to Stokes' high workloads over the series.
The workhorse captain bowled 101 overs this series, the second-most of any England bowler, on top of long stays batting at the crease.
Stokes also failed to play the final Test of the 2025 series against India with an injury in the shoulder above his bowling arm.
He was also troubled by a groin issue in 2025, notably in the series against India, and during the second Ashes Test, when he wore a ball to the area.
Stokes is not a member of England's squad for the T20 World Cup that begins next month, so should have ample time to rest the latest injury.
His next international commitments come in the Test series against New Zealand, Stokes' country of birth, in June.
Stokes' exit came just after Australian allrounder Beau Webster brought up his half-century to help Australia build on their sizeable first-innings lead.
Australia led by 149 runs when Stokes left the pitch, with vice-captain Harry Brook set to assume on-field leadership duties.
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