In skipper Andrew Crosby and Richard Hinds the home side had two excellent defenders, and with Billy Sharp proving a real thorn in the Tranmere rearguard, they proved once again they can hold their own against fancied sides.
Ian Sharps was a key figure for the Merseysiders while Sam Aiston's speed was often the cause for concern on Scunthorpe's left flank.
Scunthorpe had by far the better of the opening half with latest acquisition Sharp quickly establishing himself with a non-stop display.
He created the first opening in the 14th minute which resulted in Matt Sparrow firing straight at John Achterberg.
Five minutes later as the home side gained a free-kick, Peter Beagrie crashed a free-kick against the keeper's right-hand post from 25 yards.
Teenager Sharp then turned brilliantly and forced Achterberg to turn a shot around the post and then went close again.
Tranmere's best effort saw Chris Greenacre and Steve Davies collide on the edge of the Scunthorpe box, but the momentum remained with the home side and the keeper punched away from Beagrie.
The visitors then saw Danny Harrison fire over the top and a fine move by Aiston ended with Harrison's 25-yard shot tipped over by Paul Musselwhite.
Scunthorpe broke the deadlock in the 56th minute. Again it was Sharp who made a brilliant darting run into the box to the right before cutting the ball back to give Richard Ryan his first goal for the club on his senior debut.
Tranmere were hurt by this reverse and after going close centre-back Sharps equalised, firing into the top of the net from Aiston's ball into the box in the 61st minute.
The goals made for more open play which saw Hinds head over and a lob from Greenacre left Musselwhite struggling as it soared over the bar.
Scunthorpe grabbed the initiative again with eight minutes remaining as they created an attack down the left and as the ball was crossed into the middle, Hinds came out on top in a scramble to poke home.
In the last minute, Sharp was unlucky not to score when his curling shot struck the woodwork.