With the development of urban rail transit, the subway inevitably needs to run through the main channel of the South-to-North Water Diversion. With the subway being put into operation, the deformation during the construction period has been gradually stabilized, and the cyclic vibration load of the train during the operation period has gradually affected the channel structure. Therefore, in order to further investigate the effect of vibration load on the structure of the Middle Route of the South-to-North Water Diversion Project during the operation period, this paper, based on the actual cases of undercrossing projects in different regions, established a tunnel-soil-channel finite element model considering silty clay and fine sand under different burial depths by using ANSYS. The vertical vibration levels of the roadbed, tunnel wall and channel bottom are extracted and compared with the measured vibration acceleration levels. The dynamic displacement and maximum tensile (compressive) stress under different working conditions are analysed, and the fatigue life of the canal concrete structure is predicted using the obtained maximum tensile (compressive) stress. The results show that under the condition of fine sand, 1 times the hole diameter is the most unfavourable condition. In the prediction of concrete fatigue life by S-N equation, the number of trains that can pass under the most unfavourable condition is about 6.68 x 1010, which is far more than the number of trains that can pass within the service life of the tunnel.