To investigate the fatigue reliability of reinforced concrete (RC) bridges under stochastic traffic loading and the effectiveness of limiting the truck load, this study presented an analysis method for evaluating the bridge fatigue reliability under the action of random traffic flows based on truck weight limits. Firstly, a simply RC T-beam bridge was selected as an example to illustrate the presented approach, and the traffic-induced stress spectrum of the tensile rebars in the critical girder of the bridge was obtained. Secondly, based on the collected traffic data from two specific areas, the corresponding random traffic flow was simulated respectively, and then calibrated using the weight limit referring to the current regulation and overload violation rates ( VR) to exclude overweight trucks. Finally, based on the S–N curve from Eurocode 3 and Miner’s cumulative damage model, the effects of violation rate, average daily truck traffic ( ADTT) and truck weight limit ( TWL) on the fatigue reliability index of the bridge were studied. The results show that heavy duty trucks will induce serious cumulative fatigue damage, and degrade the durability of RC bridges, while limiting the truck weight can obviously slow down the reduction of the fatigue reliability index. Both the ADTT and the VR can significantly affect the fatigue reliability of the bridge. More specifically, the bridge fatigue reliability index shows a clear downward trend with the increase in the ADTT and the VR. Furthermore, the performance to satisfy the target fatigue reliability of the bridge was compared between TWL for each type of trucks and unified TWL for all the trucks, and then the unified TWL for all the trucks are determined to ensure the target reliability index of the RC bridge after 100 years’ service life.