Misbah smashed fifty off just 21 balls with four boundaries and as many sixes to break the 24-ball record of South African Jacques Kallis against Zimbabwe at Cape Town in 2004.
At lunch Azhar Ali was unbeaten on 72 and Misbah was 52 not out as Pakistan, 210-3 in their second innings, stretched their lead to 519 with seven wickets and five sessions left in the match.
The skipper pushed paceman Mitchell Starc for three to reach his record in 24 minutes.
The previous fastest fifty in terms of time was set by Bangladesh's Mohammad Ashraful, who took 27 minutes for his half-century against India at Dhaka in 2007.
Misbah's innings overshadowed Ali's patient knock of 139 balls with four boundaries.
Resuming on 61-2, Pakistan chased quick runs to set Australia a big target. After a 221-run victory in Dubai, the Pakistanis are looking for their first series win over Australia in 20 years.
He finished with an extraordinary tally of 474 runs in the two-match series with twin hundreds in the first match and 213 in the first innings in Abu Dhabi, a Pakistani record for two Tests.
Pakistan had scored a mammoth 570-6 declared in the first innings and then dismissed Australia for 261.
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