As the main source water supply of Xi’an city, the aquatic environment of the Heihe River directly affects the stability and sustainable development of the city and its surrounding area. To analyze the fish assemblage characteristics and historical change of the Heihe River and reveal the effect of ecologic environment change on fish assemblages, a field investigation of fish assemblage was carried out in January, May, July. and November of 2018–2019. A total of 7020 individuals from 17 species were collected, belonging to 16 genera, 5 families, and 3 orders. Cypriniformes were dominant, accounting for 82.35% of the total species. Regarding ecological guilds, fish egg sticky, omnivorous, and demersal species dominated the Heihe River, accounting for 76.47%, 64.71%, and 64.71% of the total species, respectively. Dominant species were Phoxinus lagowskii (IRI=0.5559) and Pseudorasbora parva (IRI=0.1019). The number of species (S) ranged from 12 to 17, the Shannon-Wiener diversity index (H′) ranged from 1.55 to 1.88, the Pielou evenness index (J) ranged from 0.56 to 0.66, and the Margalef species richness index (D) ranged from 1.54 to 2.15. Meanwhile, the fish biodiversity index reached its highest value in summer. Through cluster analysis and anon-metric multidimensional scaling analysis, it was shown that the fish community in Heihe River could be divided into three groups, I–III. Of these, Group I was composed entirely of fish in the Jinpen Reservoir and Groups II and III comprised fish in the canyon environment. The k-dominance curves of abundance were above either of the biomass curves, and the W-statistic values were between ?0.146 and ?0.181 in four seasons. In addition, compared with the historical data, the similarity index between 2010s and 1980s was moderately dissimilar (0.46). The results suggests that the community structure of fish species in the Heihe River, Qinling, has changed significantly. Artificial drainage system construction and habitat destruction are potential underlying reasons for the observed shifts in the fish assemblages.