The deterioration of water quality, loss of habitats, and degradation of ecosystems in urban rivers, caused by intensified urbanization, have significantly impacted aquatic biological communities, including fish. In order to establish effective conservation strategies for fish populations, it is imperative to undertake a comprehensive analysis of the relative contributions of stochastic and deterministic processes in community assembly. In this study, fish monitoring was conducted at 30 sampling sites in Guangzhou's urban rivers using both environmental DNA (eDNA) and traditional survey methods. The subsequent analysis focused on the composition and assembly mechanisms of fish communities. The results demonstrated that: (1) The fish communities inhabiting the urban rivers of Guangzhou demonstrated notable biodiversity. The eDNA monitoring survey identified 15 orders, 39 families, and 139 species, whereas the conventional survey recorded 6 orders, 10 families, and 32 species (genera). The fish assemblage was predominantly composed of Cypriniformes, Gobiiformes, and Siluriformes, with invasive species such as Coptodon zillii demonstrating ecological dominance. (2) The analysis revealed that environmental factors accounted for 49.26% and 61.15% of the observed variance in community structure, as measured by eDNA and traditional survey data, respectively. Key water quality parameters, including pH, dissolved oxygen, and the permanganate index, exerted significant effects on the restructuring of fish communities. This underscores their pivotal role in shaping community diversity and spatial distribution patterns. (3) Analysis of community assembly mechanisms indicated that deterministic processes primarily governed the assembly of fish communities in Guangzhou's rivers, while the impact of stochastic processes, including dispersal limitation, remained significant.