Accelerating global urbanization is leading to drastic losses and restructuring of biodiversity. Although it is crucial to understand urban impacts on biodiversity to develop mitigation strategies, there is a dearth of knowledge on the functional structure of fish assemblages spanning the entire city-scale spectrum of urbanization intensity. Here, using environmental DNA sampled from 109 water sites in Beijing, we investigated the taxonomic and functional diversity patterns of fish assemblages across the city and uncovered community-, trait-, and species-level responses to various environmental stressors. By ranking sampling sites into three disturbance levels according to water physiochemical and landcover conditions, we found that both native and non-native fish taxonomic and functional α-diversity decreased significantly with elevating disturbance, as strong disturbance led to the disappearance of many species. However, the quantitative taxonomic and functional β-diversity components of native and non-native fish showed distinct patterns; assemblage turnover dominated native fish β-diversity and decreased with increasing disturbance, whereas species/trait richness differences dominated non-native fish β-diversity and increased with disturbance intensity particularly in lotic waters. RLQ and fourth-corner analyses revealed that fish size, fecundity, diet, and reproductive behaviors were significantly correlated with water quality, with pollution-tolerant, larger-sized native and omnivorous non-native fishes being urban winners, which indicates strong trait-dependent environmental filtering. Potential ecological indicator species were identified based on the sensitivity of fish responses to pollution loads; these were mostly small native species, and many have bivalve-dependent reproduction. Our results demonstrate that, along with native fish assemblage simplification and homogenization, urban stressors exert profound impacts on community trait composition, highlighting the need to consider both biodiversity loss and functional reorganization in combating disturbance of aquatic ecosystems under global urbanization. Furthermore, correlations between cropland cover and water nutrient level suggested that the management of agricultural runoff might be critically important for safeguarding urban water quality.