In order to calculate the fatigue deflection of prestressed concrete (PC) beams, a fatigue variable stiffness distribution (VSD) model based on crack development pattern extracted from fatigue test is presented in this study. Firstly, a linear strain distribution model along the transfer length of concrete and steel bar was established. And the stress analysis of different cross-sections along the transfer length was performed in order to calculate the average moment of inertia in the fatigue crack zone of PC beam. Then, combined with the characterization model of concrete fatigue modulus, a fatigue VSD model for PC beams was established. The model validation shows that the calculated deflections are in good agreement with the measured deflections, and the deviation does not exceed ±10%. Furthermore, the quantitative reverse deduction of fatigue damage based on stiffness index was carried out. And the multi-stage development laws of stiffness degradation and damage evolution under fatigue loading were summarized. Finally, combined with the setting of fatigue failure threshold, the critical point of the PC beam entering the failure state can be reasonably determined. which provides a reference for the assessment of structural service status. The stiffness threshold of the fatigue failure criterion is about 70%, and the damage threshold is about 0.6 for this study. The research results can provide a theoretical basis for the structural performance analysis and failure assessment for PC beams.