In this study, the fatigue behavior of welded joints in an orthotropic steel bridge is simulated and analyzed. The traction structural stress method is proven to be more accurate and effective, and the predicted results agree well with the test results compared to traditional assessment methods, including the nominal stress, hot-spot stress, and effective notch stress methods. The traction structural stress concentration factor curves of welded joints under cyclic tensile and bending moment loading are obtained. The accuracy and validity of finite-element simulation methods for welded joints in orthotropic steel bridge are verified by comparing simulation results with full-scale tests results. Equivalent nominal stresses predicted by the master S–N curve method based on traction structural stress results agree well with the fatigue test results with small standard deviation. The fatigue behavior of orthotropic steel bridge analysis specifications Eurocode3 and GB50017 is combined with the traction structural stress method using unified traction structural stress concentration factor curves. This approach provides practical guidance for the fatigue behavior design of orthotropic steel bridges.