There is uncertainty for the cause of the abnormal seepage flow in the entry and outlet sections of the water-conveyance crossing structures. A comprehensive analysis method was proposed to explore the leakage cause in the outlet section of a cross-river aqueduct in a long-distance water transfer project in this research. First, the operation management data such as patrol inspection and reinforcement measure were analyzed. A statistical model was constructed to quantitatively analyze the relationship between the seepage pressure in the transition section and the uplift pressure beneath the sluice chamber, and the cannel water level, temperature, rainfall and other environmental variables. The main influencing factors of the seepage variables were preliminarily judged by the above-mentioned qualitative and quantitative analysis. Second, considering the engineering geology and hydrogeological conditions, the leakage cause of the protective structure could be further judged through the analysis of the surface deformation monitoring data. Then the leakage entrance and leakage channel was revealed based on several geophysical detection methods such as flow field method, ground penetrating radar and high-density resistivity method. Finally, according to the comprehensive analysis, it was found concluded that the leakage entered from the structural joints at the end of the transition section, and leaked out to the protective structure through the sand and gravel layer foundation of the transition section, the sluice chamber section and the connecting section. Considering the long-term operation of the water transfer project, it was difficult to stop water transfer for the aqueduct maintenance. Therefore, the anti-seepage reinforcement treatment plan, namely adding a grouting curtain to block the seepage channel beneath the sluice chamber, was proposed. The adopted comprehensive analysis method can provide reference for the analysis and treatment of similar engineering problems.