A steel plate-concrete composite structure reinforcement method of a structural negative moment region is characterized in that the method comprises the steps of: (1) carrying out interface treatment of a negative moment region of a concrete box girder top plate 3; (2) planting bars on the surface of the negative moment region of the concrete box girder top plate 3; (3) manufacturing a steel plate 9, a welding stud 8 and a corrugated steel plate 14: (4) forming a self-compacting concrete pouring hole 13 and a location leveling hole 17 on the steel plate 9; (5) erecting a reverse ejection support 2 and unloading reverse ejection; (6) binding a reinforcing mesh 10 and installing in position; (7) applying a structural adhesive on the steel plate 9 and spreading fine sands; (8) installing and leveling the steel plate 9 to the design position, applying a pre-stress to the top of the steel plate 9 through a pre-installed apparatus; (9) mixing and pouring self-compacting concrete; and (10) releasing the pre-stress, removing the corrugated steel plate 14, curing concrete, constructing a pavement layer, and removing the reverse ejection support 2. The novel steel plate-concrete composite structure negative moment region reinforcement method can be widely used for negative moment region reinforcement of concrete box girder, T-beam, hollow plate girder, common reinforced concrete beam, and pre-stressed concrete beam, and can be used in the bridge reinforcement with the advantages of almost no reduction of the headroom below the bridge, no requirement for surface levelness of the original structure, low cost, no influences on the appearance, and so on. The steel plate-concrete composite structure reinforcement method is economic, safe, and practical, is easy to ensure the project quality and has wide application prospect.