The Tigers, second in the table, and six points ahead of Torquay, only needed to win at Roots Hall to virtually gain automatic promotion to the Second Division.
However, Southend, who were certainties for relegation three months ago, have hit a superb run of form and goals from Mark Gower and Kevin Maher extended their unbeaten home run to ten matches.
Hull should have opened the scoring when Darryl Flahavan pushed a Ben Burgee effort to Stuart Green but the right-winger hit his shot into the side-netting.
However, Southend made the away side pay for that miss two minutes later when Hull failed to clear a Duncan Jupp cross and Gower powered in the opening goal.
But the Tigers were not behind for long as a goalkeeping error by Flahavan gifted them an equaliser. Green swept the free-kick to the back post, Flahavan came and missed the cross and Junior Lewis had the easy task to head the ball into the unguarded goal.
Just before half-time Southend's Maher had a long-range effort spectacularly saved by Boaz Myhill but the United captain was not to be denied from the resulting corner. Duncan Jupp won a 50-50 challenge on the edge of the box and the ball fell to Maher who curled a shot into the far corner with Myhill unsighted by the mass of defenders in front of him.
United nearly grabbed their third in the 53rd minute when a Lewis Hunt drive took a deflection and forced Myhill into another excellent diving save.
However, Hull equalised on 68 minutes when a wonder strike from Ian Ashbee effectively put the away side one goal away from promotion.
But they could not get that extra goal and will now be looking to beat Huddersfield Town at home on Saturday to cement their place in Division Two next season.