In a half-through steel arched bridge, the suspenders are the critical load transfer component that transmits the deck system and traffic load to the arch rib. These suspenders are subjected to traffic and environmental vibrations and are prone to fatigue failure, especially for overloaded moving vehicles. This paper aims to study the impact of moving vehicles’ overloaded rate on the fatigue performance of suspenders in a long-span three steel truss arch bridge. Based on the Mingzhu Bay steel arch bridge, a 3D finite element bridge model was first established and seven types of moving fatigue vehicle models were considered. Then the stress amplitude and dynamic response of the suspenders on the middle steel truss arch were studied under a standard, 25%, and 50% overloaded moving vehicles load. Following that, the Miner fatigue cumulative damage theory was employed to evaluate the fatigue life of the suspenders. The results show that the short suspenders in the middle steel truss arch have the shortest fatigue life but can still meet the design requirements under the standard load. However, the fatigue life of the suspenders decreases by 20% and 30% when the overloading rate reaches 25% and 50%. The fatigue life cannot meet the design requirement when the overload rate is 50%.