Due to the demand for increasing trainload and enhancing some existing heavy-haul railways, the low reserve value of bearing capacity is a problem for the 32 m-span simply supported beam. The fatigue behavior of prestressed concrete beams in a heavy-haul railway loaded by 33 t and larger axle weight of trains was experimentally investigated. The experimental results of the fatigue behaviors, including fatigue deformation, crack propagation behavior, and strains of classical materials were obtained and analyzed. A fatigue behavior assessment model was established to investigate the residual stiffness and yield point degradation of the beams loaded by the trainload. The effects of train fatigue cycles and prestress loss on the residual stiffness and yield point degradation models of the beams were analyzed. The results indicated that the crack development process had three stages during the fatigue process: the derivative stage, gradual development stage, and fatigue failure stage. Trainload was the main external factor influencing the fatigue behavior of prestressed concrete beams. The increase in trainload accelerated the degradation rate of the residual stiffness of the beams and yield point, reducing the fatigue life. The prestressing strand was primarily used to delay the concrete cracking in the tension zone. When the beam was not cracked, the prestressed concrete beam showed good fatigue performance, and the degree of prestressing did not affect the fatigue life of the beams. When the maximum fatigue load exceeded the cracking load of the beam, prestress loss in beams became a critical issue that accelerated the degradation rate of fatigue strength and reduced fatigue life. The higher the fatigue damage degree, the more pronounced the effect of prestress loss on the fatigue strength of the beams. The fatigue failure of prestressed concrete beams occurred in the bottom tensile steel bar. Therefore, when the trainload of a heavy-haul railway is greater than the cracking load of the beam, it is recommended to strengthen the beam by prestressing and strictly control the trainload to avoid yield failure.