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To evaluate the fatigue damage reliability of critical members of the Nanjing Yangtze river bridge, according to the stress-number curve and Miner’s rule, the corresponding expressions for calculating the structural fatigue damage reliability were derived. Fatigue damage reliability analysis of some critical members of the Nanjing Yangtze river bridge was carried out by using the strain-time histories measured by the structural health monitoring system of the bridge. The corresponding stress spectra were obtained by the real-time rain-flow counting method.Results of fatigue damage were calculated respectively by the reliability method at different reliability and compared with Miner’s rule. The results show that the fatigue damage of critical members of the Nanjing Yangtze river bridge is very small due to its low live-load stress level.