Objectives The aquatic germplasm resources in Longchuan River, a key area within the Yangtze River Basin in Guizhou Province, were investigated to study the diversity and distribution patterns of these resources and to provide theoretical and practical guidance for the sustainable conservation and utilization of aquatic germplasm in the region, thereby promoting the healthy development of fishery ecology in the Yangtze River Basin. Methods Water samples were collected from the Longchuan River in April 2023. Aquatic organisms were detected with fixed gill nets and multi-gear methods in May 2020, December 2022, and May 2023. Data collected on indexes of water quality, body length and weight of aquatic species, population metrics, and biodiversity index were systematically analyzed. Results The overall water quality in the Longchuan River was good, with total nitrogen as the main pollutant, accounting for 3.21 mg/L, and total phosphorus accounting for 0.062 5 mg/L. The fish resources in this section were relatively abundant. In 2023, there were 55 species of fish, 2 protected species, 9 endemic species, and 6 invasive species detected. The number of fish species (indigenous fish), the Shannon Wiener index, and the proportion of representative species in the catch of this section in 2023 was 49, 2.58, and 31.09, an increase of 44.12%, 21.70%, and 33.49% compared to that in 2020, respectively. The final scores of post-moratorium assessments of aquatic biological integrity index in the key water area of Longchuan River in Guizhou Province in 2022 and 2023 were 46.67 and 40.00, respectively. Conclusions The overall post-moratorium abundance of fish resources in the key water areas of the Longchuan River in Guizhou Province has shown an increasing trend, and the overall integrity index of aquatic organisms has remained stable. The fishing moratorium policy can play an important role in protecting the aquatic ecosystems.