This paper presents the experimental and analytical results of the fatigue behavior of prestressed concrete (PC) beam used in a heavy-haul railway under constant-amplitude and variable-amplitude fatigue loading. The fatigue behaviors including mid-span displacement, bending stiffness and strains of classical materials were investigated comprehensively. Moreover, the residual strain of concrete in compression zone was mainly analyzed and fitted, and the S-N relationships of bottom tensile steel bar and the test beam related to prestressing force were given. The experimental result shows that the fatigue failure mode of PC beam is the fatigue fracture of bottom tensile steel bar in the pure bending section. The main crack width, stiffness degradation and the maximum displacement all show development law of three stages under the constant-amplitude fatigue loading. The fatigue characteristic parameters (stiffness, displacement and strain of concrete in compression zone) of variable-amplitude fatigue test beams exhibit the multi-stage development law due to the addition of multi-level fatigue load. Further, on the basis of the fatigue characterization model of classical materials and the stress analysis of fatigue cracking cross-section, a nonlinear numerical analysis method for the whole fatigue process is proposed. The numerical simulation results are in good agreement with the test data, which shows that the proposed numerical analysis method can simulate the fatigue behavior of PC beams under constant-amplitude and multi-level variable-amplitude fatigue loading. The results of this research provide a reference for fatigue design and fatigue life evaluation of PC beams in heavy-haul railway.