Ongoing urbanization continuously reshapes water quality, habitat structure, and biological communities in river ecosystems; however, its impacts on host-associated microbial communities remain poorly documented. The fish gut microbiota, a critical interface between the aquatic environment and host physiology, is widely recognized as an integrative indicator of both environmental change and host ecological traits. This study established a continuous urbanization gradient along Shanghai’s Suzhou River, spanning from suburban areas through the outer and inner ring roads to the city center. Five common wild fish species (Coilia nasus, Hemiculter bleekeri, Culter alburnus, Acheilognathus macropterus, and Pseudorasbora parva) were collected, and their gut microbiota were characterized via high-throughput 16S rRNA gene sequencing. Significant variation in OTU richness, alpha diversity, and community structure was observed across urbanization gradients and among fish species. Principal coordinate analysis revealed that samples from suburban areas were structurally distinct from those collected in other zones, whereas inner-ring and urban-core areas exhibited substantial compositional overlap. Taxonomic analysis revealed that Firmicutes and Pseudomonadota dominated all samples; however, their relative abundances and genus-level composition varied considerably among fish species and across the urbanization gradient. PICRUSt-based functional prediction indicated that metabolic pathways predominated, particularly those involved in global and overview maps, carbohydrate metabolism, amino acid metabolism, energy metabolism, and metabolism of cofactors and vitamins. Collectively, these findings demonstrate that fish gut microbial communities exhibit spatial structuring along the urbanization gradient, with species-specific responses linked to ecological traits. This study provides valuable data on host-associated microbial communities in urban rivers and offers a reference for incorporating microbial indicators into urban water ecological assessments.