Khawaja, 28, was due to return from a hamstring injury in Thursday's opening Big Bash League match for the Sydney Thunder after being included in the 13-man Australia Test squad for Melbourne.
But Cricket Australia medical staff have ruled him out of the clash against the Sydney Sixers.
Khawaja will need to prove his fitness before Sunday's Big Bash game with the Melbourne Stars, which would be his only remaining fixture before the Boxing Day Test at the Melbourne Cricket Ground.
Khawaja has not played since injuring his hamstring in the second Test against New Zealand in Perth last month, and Australian coach Darren Lehmann said he was not willing to risk him without a warm-up game.
"We certainly need him playing and with the hamstring we don't want him playing Test match cricket if he's not fully fit," Lehmann said after Australia's innings first Test defeat of the West Indies in Hobart.
Khawaja scored 174 and 121 batting at three against New Zealand before he was injured, while his replacement, Marsh, made a strong case for his retention with his highest Test score of 182 in Hobart.
Australia captain Steve Smith is also have treatment for knee and hip injuries in the lead-up to the Melbourne Test, and has been ruled out of the Sixers' opening two Big Bash matches.
He said he expected to be fit for the Boxing Day clash.
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