In order to explore the specific causes of fastener bolt looseness and elastic strip loosening on the already built and operational rail transit lines, and to prevent similar problems from recurring on newly-built rail transit lines, this study combines with the actual situation of two rail transit elevated bridges in a certain city. Taking the failure analysis of U-beam fasteners as the breakthrough point, it explores the causes of fastener failure of ballastless track through field tests and vehicle-bridge coupling vibration simulation methods. Then, focusing on the vibration characteristics of the ladder sleeper structure and the comparison of vibration reduction and isolation effects between damper fasteners and the ladder sleeper structure, and applying the principles and methods of dynamic flexibility and energy flow, it analyzes the mechanism of factors affecting the vibration frequency of the track structure through a series of diagrams, and interprets the vibration mechanism of the track structure. The vibration effects are analyzed based on the measured and simulated data. The contribution of wheel wear to vibration is analyzed by measuring the vibration acceleration caused by vehicles in different operating years. Based on the field test, finite element simulation and vehicle-bridge coupling vibration calculation, the contribution of main girder section to vibration is analyzed. The contribution of track structure to vibration is analyzed by measuring the vibration acceleration of fasteners in different intervals; Based on the vibration field test of rail-crossing bridge, the mechanism analysis of the factors affecting the vibration frequency of rail structure is carried out with the vehicle-bridge-rail coupling vibration analysis program, and the vibration variation law is explored when the stiffness of fasteners and the stiffness of under-pillow damping pads are changed. The research results show that wheel wear, bending-torsion coupling effect of beam and vibration isolation of ladder sleeper are important potential factors that constitute fastener diseases of rail transit lines. The vibration reduction and isolation effect of ladder sleeper track structure is good, and the vibration of rail and ladder sleeper is significantly affected by the stiffness of fastener, while the vibration of bridge is significantly affected by the stiffness of damping pad. Proper reduction of fastener stiffness can significantly reduce the vibration of ladder sleeper. The research results can provide a reliable basis for improving the vibration research of rail transit bridge fastener system and provide design reference for related projects.