The Iron’s leading scorer swooped 17 minutes from time to secure victory in the Northern area final second leg at the expense of the battling Millers at Don Valley Stadium.
Scunthorpe always looked the likelier side and should have been home and dry by half-time as Hooper had a close-range shot blocked and Garry Thompson saw his effort from the rebound deflected over.
Martyn Woolford screwed a good chance wide from 10 yards in the first half and also hit the bar in the second half as Scunthorpe stayed in control.
Rotherham’s best chance fell to Reuben Reid, who had a header tipped onto the crossbar by goalkeeper Joe Murphy soon after half-time.
However the contest was settled when Hooper latched on to a through-ball before finishing coolly past Andy Warrington in the home goal.