Abstract: Under the influence of vehicular loads, the critical components of steel structure bridges are subjected to cyclic loading and fatigue, which may progressively accumulate and result in fatigue damage or even fracture. To ensure that the fatigue performance and lifespan of the track beam structure in the newly developed suspended monorail transit system meet the necessary standards, a scaled track beam model spanning the entire bridge length was constructed based on the principles of classical similarity theory encompassed by the three similarity theorems. Subsequently, fatigue testing was conducted to identify the failure points and assess the fatigue life of the track beam structure. A finite element model of the track beam structure was also established to evaluate its fatigue mechanical properties under the dynamic loading conditions of real train movements. According to the nominal stress method, the fatigue characteristics of the key joints within the track beam were determined. The findings indicate that the joints between the mid-span web and the lower flange plate, as well as the joints between the mid-span longitudinal stiffener and the lower flange plate, are particularly susceptible to fatigue. After 2 million cycles, the equivalent constant-amplitude fatigue limits for these two locations are 89.59 MPa and 70.52 MPa, respectively. The maximum stress amplitudes at the weld toes of the aforementioned joints are 38.08 MPa and 36.55 MPa, respectively, both of which are below the equivalent constant-amplitude fatigue limits, with corresponding fatigue lives of 26,056,600 cycles and 14,364,700 cycles, respectively. This suggests that the track beam structure will remain free from fatigue damage during regular operation. These research outcomes can provide a valuable reference for the analysis of fatigue stress and fatigue life of rail beam structures under moving loads in suspended monorail transit systems.