The temperature deformation of a long-span cable-stayed bridge (LSCSB) could significantly deteriorate the smoothness of rails, thereby affecting the dynamic behavior of high-speed trains (HSTs). In this paper, based on the thermal-fluid-structure coupling theory and vehicle-track coupling dynamics theory, an analysis method was proposed to explore the influence of temperature deformation of LSCSBs on the dynamic behavior of HSTs. The temperature deformation of LSCSBs within a day was analyzed, and the dynamic behavior of HSTs passing through the LSCSBs at different times was revealed. The results showed that on a summer day, the temperature deformation of LSCSBs varied significantly and rapidly, and the variation experienced a significant increase of 23.46% within just 2 h. Due to the influence of structural temperature deformation, the wheel-rail interaction, train safety, and passenger comfort changed noticeably. The construction temperature of the bridge and track decisively affected the dynamic behavior of the train. Therefore, it was crucial to construct the structure under suitable temperature conditions to prevent further exacerbation of the dynamic behavior of HSTs due to violent temperature differentials. In summary, the method proposed in this paper could contribute to a comprehensive and in-depth understanding of the operational characteristics of HSTs on LSCSBs.