For some long span cable supported bridges, the longitudinal diaphragms in main girder are designed as steel truss to reduce structural self-weight. However, unanticipated fatigue cracking in critical details of longitudinal steel truss diaphragm may occur after only a few years of service, resulting in stiffness weakening and stress redistribution. Based on an actual cable-stayed bridge, this paper presents a fatigue analysis of longitudinal steel truss diaphragms and provide an effective strengthening measure to elongate the fatigue life. Using traffic information from bridge toll station, the fatigue vehicle models were established. Then, a multi-scale finite element (FE) model was developed to help determining critical details of potential cracking and calculating the vehicle induced stress. After obtaining the required parameters, fatigue life of the specific critical detail was estimated base on damage accumulation law. The result agrees well with field observation. To ensure the performance of steel girder, the strengthening measure that replace diagonal tubes in longitudinal steel truss diaphragm with bolted channel steels was proposed and then applied. The assessment result indicated that the provided strengthening measure achieves satisfactory effects. It can also provide experience and partial reference for maintenance of similar structures.