The concept of in vitro prestressing originated from France at the earliest and is one of the important branches of post-tensioning prestressing system, which usually adopts large-diameter steel bars, steel strands and high-strength steel wires as tension application tools to prestress the beams. This method can effectively reduce the stress level of the structure and can play the roles of reinforcement unloading and changing the internal force distribution of the structure, and at the same time can improve the load bearing capacity, crack resistance and stiffness of the structure. However, the in vitro prestressing method is less studied in reinforced steel truss structures, and the related theory is relatively lacking. In order to study the effect of in vitro prestressing with different number of strands on the strengthening of steel truss bridges, this paper takes a 128 m span under-bearing simply-supported steel truss bridge over the Omo River as the research object, and proposes three in vitro prestressing strengthening schemes for its load lifting analysis, and uses finite element software to establish the full bridge model with 7, 9, and 11 strands per beam respectively, and analyzes the strengthening of steel trusses in terms of rod strength, structural stiffness, fatigue, overall stability, and nodal plate stress distribution. The result analysis shows that:the stress of longitudinal beam, crossbeam, top chord and bottom chord gradually decreases with the increase of the number of strands, and the stress of the bars gradually improves; the number of strands increases, the deflection of the structure gradually decreases, the fundamental frequency gradually increases, and its stiffness continuously improves; with the increase of the number of strands used for reinforcement, the other strands, except for the longitudinal beam, gradually increase. With the increase in the number of strands used for reinforcement, in addition to the longitudinal beam, the fatigue stress amplitude of the rest of the rod is constantly reduced; the critical load factor is constantly increased, and the stability is increased; the area of the low stress area of the important parts of the node plate is increased, and the area of the high stress area and the secondary high stress area is reduced, and the number of screw holes in the potential tearing area is constantly reduced, and the probability of tearing damage of the node plate is reduced. However, as the number of strands increases, the axial force of the lower chord increases and the stability safety factor of the section decreases, so it is necessary to control the number of strands tensioned and limit the magnitude of load lifting. The results show that all the three in vitro prestressing reinforcement solutions can achieve the load lifting effect, and with the increase of the number of strands used, the structural strength, stiffness and stability are non-linearly improved, and the proportion of low stress distribution area of the node plate is gradually increased, and the bridge is reinforced by 11 single-steering bending line shaped in vitro prestressing strands, which is the most practical and reasonable reinforcement solution.