The midfielder fired in off the bar midway through the second half moments after Rob Hall hit the Millwall post.
Earlier, Millwall's Lee Gregory saw a penalty appeal turned down and Angel Martinez volleyed wide from six yards before the break.
The Lions have one win in eight and sit 20th, six places and three points behind Bolton.
Wanderers' latest win included a fourth-straight clean sheet and maintained their impressive run of form since Neil Lennon became manager on 12 October.
Since then, they have jumped from bottom spot in the table to 14th with just two defeats in 11 games.
At The Den, their victory was richly deserved as though Millwall rallied late on, the Trotters managed 14 shots to their hosts' eight.
Eidur Gudjohnsen was handed a first start for Bolton since 2000 after agreeing a return until the end of the season.
His team-mates Pratley and Tim Ream worked home goalkeeper David Forde in the early exchanges, either side of a glorious opening for Gregory, who shot wide of the Bolton goal after collecting a long ball.
Magaye Gueye fed Andy Wilkinson to deliver accurately for Martinez to miss the target with his volley before the break, but it was moments after the restart when the game's contentious moment arrived.
Again it was Gregory - the matchwinner in Millwall's 1-0 win at Brighton last week - who tested the visiting defence and he appeared to be pulled down by Bolton goalkeeper Andy Lonergan but referee Stuart Attwell waved away appeals.
If given, the penalty would likely have seen the keeper sent off but his side capitalised when Pratley raced on to Chung-Yong Lee's pass to steady himself and fire a second goal of the season.
Lonergan denied Martyn Woolford with a smart save in the closing stages as Bolton held on for a second away win of the season.
Bolton extended their unbeaten run to seven games with Darren Pratley's second-half winner at Millwall.