Structural health monitoring (SHM) system provides valuable information for the fatigue assessment of existing rail-cum-road bridges. This paper aims to develop an effective fatigue assessment approach for the rail-cum-road bridge, Jiujiang Yangtze River Bridge, by utilising the SHM data, focusing on dynamic modal analysis, finite element model updating and fatigue assessment. First, the traffic load spectrum and modal characteristics of the bridge are investigated from the SHM data. A three-dimensional finite element model is constructed and then updated by using the measured modal data through the proposed regularised model updating method. Then, the updated numerical model is verified with the measured dynamic response data, which can be utilised for calculating stress response at critical structural components of the rail-cum-road bridge. Finally, an improved Corten-Dolan’s model is proposed to analyse the fatigue damage and structural reliability of the critical structural components of the bridge, taking into account the combined effects of train and highway vehicle loads. The results demonstrate that the proposed fatigue assessment method provides more reliable results for the rail-cum-road bridge by considering the combined effect of multi-level traffic loads and the non-linear fatigue damage accumulation. It is concluded that the short H-shaped suspender is identified as the most vulnerable structural member of the rail-cum-road bridge, and the remaining fatigue service life of the typical components of the bridge should meet the design requirement.