Goals from Manchester United loanee Ashley Fletcher plus Adam Hammill and Lloyd Isgrove saw off the Lions, who had halved an early deficit through boyhood Barnsley fan Mark Beevers.
It caps a remarkable 2016 for Paul Heckingbottom’s men, who were bottom of League One on December 11 but went onto win the Johnstone’s Paint Trophy before securing their place in the play-offs by finishing sixth.
Millwall’s preparations suffered big disruption in the warm-up with Byron Webster breaking down injured, which meant the Lions’ back four had to be reshuffled with Tony Craig drafted in as a centre-back.
It took time for Millwall to adapt to their new-look defence and Barnsley took full advantage inside two minutes.
Sam Winnall flicked the ball into the path of Fletcher, who took the ball in behind Craig and fired into the bottom corner.