Abstract: In recent years, environmental DNA metabarcoding (eDNA metabarcoding) has been widely utilized in fish diversity assessments due to its cost-effective and non-invasive strategies with increased sensitivity. To explore new methods for monitoring and protecting Erhai Lake’s fish diversity, this study first used eDNA metabarcoding to detect fish diversity of Erhai Lake. This study used a standardized process of eDNA metabarcoding analysis, including water collection, water filtration, eDNA extraction, genetic marker amplification, sequencing and bioinformatic analyses. A total of 17 fish species were detected from 9 sampling sites, including 5 native species and 12 non-native species. Five species (Carassius auratus, Hypophthalmichthys nobilis, Pseudorasbora parva, Misgurnus anguillicaudatus, and Gambusia affinis) were the dominant species. Although eDNA metabarcoding cannot completely replace traditional methods, it can be used as a supplementary tool to efficiently assess and monitor fish diversity and species distribution patterns in Erhai Lake.