The present study aimed to investigate macroinvertebrate assemblage in relation to water quality and habitat availability along the longitudinal gradient of an urban Apies River in Pretoria, South Africa. Macroinvertebrate and water sampling were conducted during dry and wet seasons, from December 2019 to March 2020, and between February 2021 and December 2022 at the three sites in the Apies River. The water exhibited neutral to slightly alkaline pH in all sites throughout the study, whereas significant differences were observed for nutrients and sulphate levels between the three sites. The nutrient levels exhibited oligotrophic, eutrophic, and mesotrophic conditions at Sites 1, 2, and 3, respectively. Moreover, the macroinvertebrate assemblage showed an association with habitat scores and water quality. The diversity was relatively higher at Sites 1 and 3 compared to Site 2. However, sensitive taxa were associated with Site 1, whereas tolerant taxa were associated with Sites 2 and 3. The average score per taxon ranged from 4.88-5.68, 3.27- 4.07, and 3.83-4.61 at Sites 1, 2, and 3, respectively. The findings of this study highlight the importance of urban Apies Rivers in providing habitats for aquatic biota and the role of anthropogenic litter in shaping macroinvertebrate community structure.