During the service period of a prestressed concrete bridge, as the number of cyclic loads increases, cumulative fatigue damage and prestress loss will occur inside the structure, which will affect the safety, durability, and service life of the structure. Based on this, this paper studies the loss of bridge prestress under fatigue load. First, the relationship between the prestress loss of the prestressed tendons and the residual deflection of the test beam is analyzed. Based on the test results and the main influencing factors of fatigue and creep, a concrete fatigue and creep calculation model is proposed; then, based on the static cracking check calculation method and POS-BP neural network algorithm, a prestressed concrete beam fatigue cracking check model under repeated loads is proposed. Finally, the mechanical performance of the prestressed concrete beam after fatigue loading is analyzed, and the influence of the fatigue load on the bearing capacity of the prestressed concrete beam is explored. The results show that the bridge prestress loss characterization model based on the POS-BP neural network algorithm has the advantages of high calculation efficiency and strong applicability.