Semi-arid watersheds are highly sensitive to external disturbances due to high evaporation rates and uneven precipitation distribution. The mechanisms and responses of aquatic biological integrity to hydrological processes across different spatial scales remain poorly understood. This study focuses on the Wei River Basin, a typical semi-arid watershed in China, identifying three hydrological variability patterns based on daily flow magnitude, coefficient of variation, skewness, and baseflow index. A coupled approach combining Generalized Linear Mixed Models (GLMM) and Piecewise Structural Equation Models (pSEM) was developed to analyze the hydrology-aquatic biological integrity responses in semi-arid watersheds. This approach quantitatively revealed the response mechanisms of hydrological variability on the integrity of phytoplankton and invertebrates in different zones of the Wei River Basin. The results showed that: (1) The spatial differentiation patterns of high variability zones (HIZ), medium variability zones (MIZ), and low variability zone (LIZ) were identified, highlighting strong spatial similarities between hydrological rhythm changes and aquatic biological integrity across the basin. (2) There is a positive correlation between the explanatory power of hydrological variability intensity and aquatic biological integrity indices. The impact pathways differ among hydrological variability zones, with hydrological variability primarily influencing aquatic biological integrity indirectly by regulating water quality and physical habitats (e.g., substrate composition) rather than directly altering runoff processes. (3) In semi-arid watersheds, phytoplankton integrity is more sensitive to hydrological variability and its spatial scale, with limited influence from seasonal random effects, whereas invertebrate integrity is more affected by seasonal random effects. Overall, hydrological variability significantly shapes aquatic ecosystem integrity in semi-arid watersheds through its combined spatial and seasonal effects.