To clarify the characteristics of macrobenthic assemblages and their response to the aquatic environment in the upper and middle reaches of the Heihe River, water quality, sediments and macrobenthos measurements were conducted in the summers of 2018 and 2019. The results showed that 50 species of macrobenthos were identified, belonging to 3 phyla, 7 classes, 15 orders and 32 families, mainly arthropods (37 species) and mollusks (11 species). Argyroneta aquatica, Chlaznius sp., dragonfly nymphs, Palaemon modestus, Radix auricularia, Cyraulus albus and Suecinea sp. were the dominant species in the whole study region; most of these are pollution-tolerant and moderately pollution-tolerant species. The macrobenthos density and biomass ranged from 10 to 577 ind./m² and from 0.0907 to 50.0562 g/m², respectively, showing high spatial heterogeneity. Predators were the main functional feeding group. One-way analysis of variance clarified that Margalef's index and the Shannon–Wiener index differed significantly among the spatial areas (P < 0.05). Canonical correspondence analysis showed that the spatial heterogeneity of the macrobenthos was affected by the water temperature and the total nitrogen and total phosphorus in sediments (P < 0.05).