Gambhir, 27, hit seven fours and a six in his 104-run knock and put on 182 runs with Virender Sehwag (90) to take India's overall lead to 431 after resuming on 100 without loss.
At the break, Mahendra Dhoni was batting on 26 after promoting himself up the order and Sourav Ganguly on three on a Punjab Cricket Association wicket that still played easy.
Sehwag, who turned 30 on Monday, struck eight fours in his 122-ball knock while Gambhir also punished the bowlers, reaching his second Test century with a four off leg-spinner Cameron White.
It was thrilling morning session for the hosts, who made 469 all out in their first innings before bowling Australia out for 268, with Gambhir and Sehwag scoring at nearly five runs an over.
White (1-30) was forced out of the attack after he was hammered for 13 runs in an over. Sehwag scooped White over mid-off for a four before Gambhir despatched the ball into the stands with a six over long-on.
The openers also rode their luck after Gambhir was trapped leg before by Peter Siddle off a no ball and Sehwag appeared to edge Mitchell Johnson but the appeal was turned down by umpire Asad Rauf. TV replays suggested a faint edge.
Sehwag, then on 88, was out after adding four more runs when a thick edge off Peter Siddle (1-62) was taken by Brad Haddin behind the stumps.
Gambhir's knock was cut short by White when he had him caught by Michael Hussey at mid-off.
The Mohali Test will be followed by matches in New Delhi (Oct 29-Nov 2) and Nagpur (Nov 6-10).