Abstract: Liujiang River is the second largest tributary of Xijiang River in the Pearl River. It has rich species diversity and complex habitat types, which play an important role in maintaining the biodiversity of the basin. In aquatic ecosystems, fish species richness and assemblages have been recognized as one of the most sensitive and reliable indicators for ecosystem processes and function. Maintaining fish diversity is the basis of the sustainable development of fisheries. To understand the diversity of fish community structure and its relationship with environmental factors in the Liujiang River, fish and environmental surveys were conducted in 9 sections of Liujiang River (Rongjiang, Congjiang, Rong’an, Liucheng, Xiangzhou, Yongfu, Luzhai, Hechi, and Yizhou) in both July and December 2018. A total of 36474 individuals belonging to 115 species, 80 genera, 20 families, and 5 orders were collected, among which, Cyprinidae was the most abundant with 68 species, accounting for 59.13%. Shannon-Wiener diversity index, Simpson index, Pielou evenness index, and Margalef richness index ranged from 2.13 to 3.12, 0.81 to 0.93, 0.58 to 0.80, and 4.65 to 7.18, respectively. The fish diversity was relatively rich, the fish richness was high, and the individual distribution was relative uniform. The dominant fish species were small fishes, such as Squalidus argentatus, Sinibrama macrops, Microphysogobio chenhsienensis, Xenocypris macrolepis, Pseudohemiculter dispar. The abundance/biomass comparison curve showed that the fish communities in Yizhou, Rongjiang, Luzhai, Liucheng, Xiangzhou, and Rong’an were moderately or seriously disturbed, and there were relatively stable in Hechi, Congjiang, and Yongfu. The spatial and temporal distribution characteristics of fish communities were analyzed by non-metric multidimensional scale sorting (NMDS), and the fish communities were divided into three groups: Congjiang-Rongjiang group in the upper reaches of the Liujiang River, Yongfu-Luzhai group in the tributaries of the Luoqing River, and Rong’an-Liucheng-Xiangzhou- Hechi-Yizhou group in the middle and lower reaches of the Liujiang River and the tributaries of the Longjiang River. Canonical correspondence analysis (CCA) was used to analyze the relationship between the fish community and environmental factors. It was found that river width, altitude, and pH were the main impact factors of fish community structure in the Liujiang River. Compared with the historical data, the fish community structure in the Liujiang River has been changed significantly, the proportion of economic fish has decreased, and the fish individuals have become smaller and younger. In addition, the Liujiang River was threatened by alien aquatic species invasion. There are several ways to strengthen the protection of fish diversity in Liujiang River such as strengthen law enforcement, crack down on illegal activities such as poaching and electric fishing; released the economic fish; restoration of river connectivity; to monitor, rescue and breed the rare and endemic fish in the basin.