As the largest lake in South China, Wanlv Lake is also an important drinking water source for Guangdong and Hong Kong and is responsible for water supplying of more than 40 million people in the Guangdong-Hong Kong-Macao Greater Bay Area. The study of fish diversity in Wanlv Lake can help us to effectively understand the changes and states of its aquatic ecosystem and provide scientific basis for natural ecological protection, biodiversity, sustainable use and scientific management of fishery resources. However, research on the lake’s fish is scarce. The high throughput environmental DNA metabarcoding technology (eDNA technology) of the mitochondrial 12S rRNA gene sequence was herein used to conduct a preliminary exploration of Wanlv Lake fish diversity. A total of 10 sampling sites were set up to monitor fish diversity and analyze the composition and richness of the species. The results revealed a total of 83 genera in 42 families and 17 orders of fish in the lake, with the highest proportion found in Cypriniformes (38 species), accounting for 45.24% of the total. Five alien species and eight rare and endangered species were also detected. The proportion of invasive species was 5.95%, and the combined proportion of rare and endangered species was 9.52%. The results indicated that Wanlv Lake serves as an important drinking water source, and overall, the condition of fish is satisfactory. However, proactive measures should be implemented to control the overpopulation of invasive species, as their unchecked proliferation may lead to a decline in species abundance, particularly among endangered species. This was the first overall assessment report on fish of the Wanlv Lake via 12S rRNA; the results herein lay a foundation for water quality assessment of Wanlv Lake as a drinking water source.