Understanding impacts of rapid urbanization on structure of river fish communities is the key basis for scientific management and biodiversity conservation for urban rivers. We selected two representative basins with different degrees of urbanization in the typical urbanized city, Shenzhen City, China. Field sampling was conducted in both wet (August) and dry (November) seasons in 2019. We compared and analyzed the differences of fish communities between the two basins using the multiple statistical methods, and explored the key environmental factors driving the variations of fish communities. Results showed that species composition, dominant species, and biodiversity of fish in the highly urbanized Guanlan River Basin were significantly different from those in the less urbanized Pingshan River Basin. In particular, the sensitively indigenous fish species such as Schistura fasciolatus and Parazacco spilurus disappeared while the invasive tolerant species were the dominant species in the highly urbanized basin. Fish biodiversity was significantly lower in the highly urbanized basin than the less urbanized basin (P<0.05). Meanwhile, non-native fish species had spread rapidly in urbanized rivers of Shenzhen City, which accounted for more than an average of 92.5% in the urbanized river sites. For environmental factors, total nitrogen, total phosphorus, ammonia, chemical oxygen demand, biochemical oxygen demand, and permanganate index in the highly urbanized Guanlan River Basin were significantly higher than those of the less urbanized Pingshan River Basin (P<0.05). Redundancy analysis based on the Bray-Curtis distance showed that the proportion of urban land and total nitrogen were the main factors affected the difference of fish communities in the Guanlan and Pingshan River Basins. Changes of river habitat from urbanization reduced biodiversity of the native fishes. Thus, restoring native fish biodiversity in river ecological management and protection is the important direction of aquatic objective-based management.