Rapid global urbanization poses considerable ecological risks to freshwater systems, notably leading to substantial reductions in microbial communities. To assess the impacts of human activities on these communities, we applied the high-throughput amplicon DNA sequencing to examine spatial variations in riverine microbial communities within an urbanized watershed. Coupled with the Geographical Detector Model, the effects of the land use were identified across the watershed. Results show that microbial communities were closely linked to the human-impacted land use patterns. The upstream region, dominated by forest cover (71%), exhibited the highest microbial population (3384 OTUs), whereas the urbanized downstream outlet (91% urban land) showed the lowest microbial population (471 OTUs). Additionally, the spatial distribution of the human-impacted land use appears to abruptly alter microbial pathways along the river. The spatial threshold effect of human-impacted land use is indicated by a Moran's I value exceeding 0.80. Notably, a 300-m buffer zone around different land uses seems to significantly influenced sediment microbial communities. Besides, the influence of land use on microbial communities is intensified by spatial drivers. For instance, agricultural land use was found to impact riverine Parcubacteria communities, with factor detector values increasing by over 30% in 400–500 m buffer zones. These findings provide new insights into the complex relationship between human activity and riverine microbial communities, highlighting important implications for ecosystem management in rapidly urbanization regions.