Australia lost David Warner for 36 and Marnus Labuschagne, run out for duck, in Saturday's first session at Karachi's National Stadium.
Khawaja is unbeaten on 52 from 83 balls and Steve Smith is not out seven, after Australia won the toss.
Khawaja and Warner put on 82 runs — an Australian record opening stand in Tests in Karachi — from 18 overs.
But Warner, after hitting two sixes and three fours in his 48-ball innings, fell to Faheem Ashraf (1-20), who missed the drawn series-opener after testing positive to COVID-19.
The Pakistan right-armer, bowling around the wicket, angled a delivery into Warner which moved away off the seam, nicking the outside edge to present wicketkeeper Mohammad Rizwan a simple catch.
Khawaja, after his 97 in the first Test, soon posted a flowing half-century, striking seven fours and a six to reach the milestone from just 69 balls.
But his union with Labuschagne lasted only three overs before the Australian No.3 was run out by a superb throw from Sajid Khan.
Labuschagne drove spinner Nauman Ali to mid-off and set off for a single but Sajid swooped and launched a side-arm throw which struck the stumps with the Australian just short of the crease.
Labuschagne's dismissal left Australia 2-91 as the visitors seek to redress their poor record in Karachi, where they have five losses and three draws from eight Tests.
The venue is a favourite for Pakistan, who played 34 Tests there before losing — overall they have 23 wins, 18 draws and two defeats at the National Stadium.
The hosts recalled Faheem and paceman Hasan Ali, who missed the dull draw in Rawalpindi because of a groin injury. The duo replaced Naseem Shah and Iftikhar Ahmed.
Australia have given legspinner Mitchell Swepson a Test debut at the expense of fast bowler Josh Hazlewood.
The 28-year-old Swepson, who was first in an Australia's Test squad in 2017, was presented his baggy green cap before play by his Queensland captain Khawaja.
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