Benthic diatom assemblages can sensitively respond to aquatic environmental changes. However, it is still ambiguous whether benthic diatom assemblages can well indicate the environmental status in an urban river with highly heterogeneous habitats. This study investigated the environmental heterogeneity of an urban river and explored the response of benthic diatom assemblages to environmental changes. First, the urban river showed significant spatio-temporal heterogeneity in both physical and chemical characteristics. Higher concentrations of total nitrogen and CODMn indicated eutrophic and organic pollution conditions in the river. In addition, benthic diatom assemblages also implied the habitat heterogeneity of the urban river. Dominant diatoms, including Nitzschia palea, Melosira varians, Cyclotella meneghiniana, and Achnanthidium minutissimum, are pollution-tolerant species, further confirming the eutrophic and organic status. Then, the interaction between environmental heterogeneity and benthic diatom assemblages was analyzed by redundancy analysis and Mantel tests. Results suggested that environmental variables, such as conductivity, total nitrogen, total phosphorus, and chlorophyll a, significantly affected the composition of benthic diatom assemblages in the urban river. These findings demonstrated that environmental heterogeneity shapes the benthic diatom assemblages of the urban river, and benthic diatoms can be good indicators of environmental status for urban river monitoring and assessment.