Abstract: Yuzhuo Reef is an important atoll in the Xisha Islands, with diverse coral reef ecosystems and rich fish resources. In order to understand the status of coral reef fish resources in Yuzhuo Reef and their evolutionary features, we investigated the fish resources of Yuzhuo Reef in 2023 through methods such as handline fishing, underwater spearing, underwater videography and environmental DNA (eDNA) analysis, identifying 220 fish species in total, of which 111 were discovered through the eDNA method alone. Combining with the historical archival data from the South China Sea Fisheries Research Institute in 2003, we had already recorded a total of 265 fish species in Yuzhuo Reef, which belong to 12 orders, 50 families and 128 genera, with Perciformes species dominating the species composition. The fish community composition was primarily characterized by small-size fish, with the average taxonomic distinctness index (Δ⁺) and variation in taxonomic distinctness index (Λ⁺) being 56.08 and 122.4, respectively. Similarity analyses show that from 2003 to 2023, the similarity in species composition, dietary habits and body size was either dissimilar or extremely dissimilar. Compared with the historical records, 45 fish species were not observed; particularly large carnivorous fish were most significantly affected by natural and human disturbances. Conversely, the impact on phytophagous fish was relatively small, with siginificant decline in taxonomic diversity indexes (Δ⁺, Λ⁺). It is suggested that unsustainable fishing practices, habitat degradation and rising temperatures have contributed to changes in the fish community structure in Yuzhuo Reef. The findings provide references for the conservation and management of coral reef fish resources and the rehabilitation of coral reef ecosystems in Yuzhuo Reef.