To understand the fish diversity and assemblage structure in the middle and lower reaches of the Yiluo River and to assess the impacts of human activities on river ecosystems, six fish sampling occasions in five sites were conducted bimonthly in 2016. A total of 12361 individual fishes were collected in the river stretches, comprising 43 species belonging to 4 orders, 9 families and 37 genera. The Cyprinidae family, representing 28 taxa, accounted for 65.1% of the total number of fish species collected. Shannon-Wiener diversity index, Margalef species richness index, Pielou evenness index and Simpson's diversity index of fish community in the investigation regions ranged from 1.75 to 2.38, from 2.44 to 3.63, from 0.59 to 0.76 and from 0.73 to 0.86, respectively. Fish species diversity in Xicaodian reach was highest among the five reaches. Abundance-Biomass Comparison (ABC) Curve showed that all sampling sites were subject to varying degrees of interference apart from Xicaodian reach. Fish communities in Qilipu reach and Heishiguan reach were subject to the maximum interference with the dominant fish species were mainly small-sized fish or young individuals of large-sized fish. Redundancy analysis was used to describe the relationship between the assemblage structures and associated environmental factors. Ammonia nitrogen, total phosphorus and pH accounted for the largest amount of variance. In view of the status of fish species diversity in the middle and lower reaches of the Yiluo River, it was suggested that managers should take steps, such as strengthening water quality management, restoring river connectivity, fishing banning in the reserves and conducting habitat restoration, to ensure the ecological protection and river health.