In response to the complex fluid-structure interaction effects of water sloshing in thousand-tonnage navigation aqueducts located in high-intensity seismic zones of southwestern China, this study takes the second-line ship lock aqueduct of the Wujiang Pengshui Hydropower Station as the engineering background to investigate the characteristics of water sloshing in the tank under seismic action and its influence on structural response. Based on the ANSYS platform, a 1:1 three-dimensional finite element model of the aqueduct–water coupling system was established. Using a separated two-way fluid-structure interaction solution method combined with seismic acceleration time-history inputs, the study analyzes the laws governing free surface fluctuations, transverse flow velocities, and structural stress responses under different peak ground accelerations and water depths. The results show that as the peak ground acceleration of the seismic wave increases, the wave height of the liquid surface in the tank exhibits an approximately linear growth trend. Increasing water depth significantly enhances both the amplitude of water sloshing and the characteristics of sustained oscillations. The free surface fluctuations exhibit large-amplitude anti-phase oscillations at both ends of the aqueduct, while the wave height remains relatively stable in the mid-span region, where transverse flow velocity fluctuations are most significant. Further comparison of structural responses between empty and water-filled aqueduct conditions reveals that water sloshing alters the characteristics of the structural stress distribution.