Using the macroinvertebrates collected from mountain and lowland ecoregions in the southern basin of Wei River as indicators, we compared the differences in the composition of 29 macroinvertebrate trait categories and seven traits and functional diversity indices between the two ecoregions. The combination of the joint analysis of R (environmental×sites), L (species×sites), and Q (trait×species) (RLQ) and fourth-corner analysis methods was conducted to explore the relationships between macroinvertebrate trait composition and land use and physical and chemical variables. We then used a generalized linear model (GLM) to compare differences in the responses of macroinvertebrate traits and functional diversity to environmental variables at different spatial scales between the two ecoregions. Our results showed that 18 of the 29 biological traits and 6 of the 7 traits and functional diversity indices significantly differed between the two ecoregions. Macroinvertebrates with biological traits such as none and shelters of leaf parts or wood refuge, lightly sclerotized, well protected exoskeleton or external protection, herbivores, and predators preferred habitats in the mountain ecoregion, while biological traits with integumentary respiration, soft body, and collector-gatherers tended to frequently occur in the lowland ecoregion under higher intensity of human activities. All traits and functional diversity indices, except functional evenness, were significantly higher in the mountain ecoregion than in the lowland ecoregion, which demonstrated that environmental stressors significantly decreased the biological traits and functional diversity in lowland areas. The combination of RLQ and fourth-corner methods revealed that the traits demonstrated a predictable response of trait patterns along the environmental gradient. The GLM model predicted that the functional and trait diversity in mountain and lowland ecoregions could be explained by different environmental variables at different spatial scales, with catchment urban land use, water temperature, and total nitrogen explaining most variations within the biological traits and functional diversity in the mountain ecoregion; but with reach scale agriculture land use and average water depth mainly explaining the decrease of the functional diversity in the lowland ecoregion.