In river ecosystems, environmental filtering selects for aquatic biological functional traits that exhibit specific responses to catchment-scale land use patterns. To elucidate the mechanisms by which spatial land use configurations influence the distribution of aquatic biological functional traits, this study conducted systematic surveys at 30 sampling sites in the Chishui River Basin from 2022 to 2024. Four macroinvertebrate functional groups were identified through functional trait matrix analysis and cluster analysis. Significant traits for each functional group were screened using Gini coefficients derived from random forest models. Multiple linear regression analysis was employed to examine the variation in explanatory power (R2) of habitat factors within buffer zones of 50, 100, 250, 500, 1000, and 2000 m. Based on the principle of R2 maximization, a 500-m circular buffer zone was determined as the optimal spatial scale for each sampling site. Generalized additive models were subsequently applied to identify the response intervals of buffer zone land use structure that maintain stability of each functional assemblage: forest land 15.21%–44.89%, cropland <5.29%, built-up land 1.21%–9.00%, bare land >32.49%, and water body 0.64%–10.89%, along with critical habitat parameter ranges (water temperature 15–24℃, pH 5.02–8.81, NH?-N 0.31–0.45 mg/L, substrate particle size 36–188 mm). This study further quantified the effects of natural land proportion on the suitability of each functional group: a 10% increase in natural land corresponded to suitability increases of 0.33 and 0.34 units for scrapers and predators, respectively; filters and collectors also exhibited significant suitability improvements with elevated natural land proportion. These findings reveal the response relationships and underlying mechanisms among land use, habitat factors, and macroinvertebrate functional groups, providing quantitative foundations for catchment ecological conservation and land management.