Stokes returned later in the day at the SCG - batting low down at No.8 - but looked to be in discomfort and was dismissed for one run.
The superstar allrounder lasted less than 15 minutes on Wednesday morning before pulling up sore midway through bowling an over.
A disappointed Stokes, who has a recent history of groin injuries, hobbled off the pitch to applause from the crowd.
Stokes was assessed for an injury to his adductor muscles and played no further part in England's innings in the field.
England bowled Australia out for 567 during the first session and he came out to bat later but was clearly unable to run.
Stokes nicked Beau Webster's off-spin to Steve Smith in the slips on the fifth ball he faced.
"He's moving pretty gingerly," said Jacob Bethell, who top-scored for England with an unbeaten 142.
"When he was out in the middle, he just said he'll be walking them. It's not going to be one (run) unless it's a long one. I don't think that bodes too well for him bowling tomorrow."
Stokes's injury came after teammate Bethell conceded on day three that the seven English players who had featured in all five Tests this summer were 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's high workloads in the series.
The workhorse captain bowled 101.1 overs this series, the second-most of any England player, on top of long stays at the crease as a batter.
Stokes also failed to play the final Test of the 2025 series against India with a shoulder injury above his bowling arm.
He was 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 and will have ample time to rest his latest injury.
His next international commitments come in the Test series against New Zealand, Stokes's country of birth, in June.
Vice-captain Harry Brook assumed the on-field leadership duties in Stokes's absence during the final Ashes Test.
England finished day four 119 runs ahead of Australia, going to stumps at 8-302.
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