Tom Hopper’s first-half tap-in and Paddy Madden’s 20th goal of the season were the difference on a sunny afternoon at Bramall Lane.
Despite victory Scunthorpe were forced to settle for seventh place after Barnsley’s resounding win at Wigan clinched the final play-off spot.
Nigel Adkin’s side finished in a desperately underwhelming 11th.
The Blades came firing out of the blocks with Alex Baptise’s brilliant header forcing Luke Daniels into a superb save sprawling low to his right.
Scunthorpe’s first effort of the game came as Luke Williams and Hakeeb Adelakun combined on the edge of the Blades’ box with the former hitting straight at George Long.
But it was the visiting Iron who took the lead six minutes before the break.
Blades’ stopper Long foiled Madden’s attempt to round him but could only parry into the path of Hopper who swept home the opener.
Four minutes later Adelakun came close to doubling the lead after some trickery got him into the box, with Long on hand to keep him out.
The Blades’ best chance to draw level fell to hit man Billy Sharp who produced a fine stop low to Daniels left from range.
Irishman Madden put the game to bed just after the hour mark as his deflected effort from 20 yards nestled in the back of the net.
With two minutes remaining Ben Whiteman curled just wide of the post as he tried to pull back a late consolation.