Chongqing Chaotianmen Yangtze River Bridge is the world's longest-span arch bridge. The node connections on its main truss’s lower chords are to bear millions of large-amplitude stress cycles caused by automobile and light-rail train loads. However, there exists no referential designing and testing experience in such detailing on long-span steel bridges at home and abroad as yet. High-cycle fatigue behavior testing on a detailing model has been conducted to study the fatigue reliability of the bridge’s critical node connections. The testing cycling load on the model is determined according to conventional specifications and the expected traffic flow on the Bridge. The 1/2-scale detailing model is carefully fabricated to simulate the node prototype and undergo the 2 million-cycle testing with the design load. Through testing, fatigue reliability of Chaotianmen Yangtze River Bridge’s main-truss lower-chord node connections during the design service life is verified. In addition, static test data are compared with those calculated by finite element analysis to prove the validity of the node model. In the end, fatigue destruction test of the model was carried out to get the fatigue failure law of the node connection and provide necessary parameters and reference for designing and monitoring of this bridge in the future.