Environmental DNA (eDNA) technology is a non-invasive, highly sensitive, efficient and non-destructive to the environment, no damage to the organism investigation tool. To select suitable metabarcoding primers for freshwater mussel's environmental DNA biodiversity research, 11 pairs of primers (with 9 pairs of universal primers designed and 2 pairs cited from relevant literature) were screened by general amplification and high-throughput sequencing of genomic DNA of 24 common mussels in the Lake Poyang Basin. The results showed that the primer cyt b and 16S rRNA had good amplification effect and high discrimination. The two pairs of primers were further verified with environmental samples (n=6) and traditional sampling. It was found that 8 mussels species belonging to 6 genera were annotated by 16S rRNA which accounted for 26.69% of the total sequences, and 5 mussels species belonging to 4 genera were annotated by cyt b which accounted for 6.60% of the total sequences. The primer 16S rRNA was more suitable used for metabarcoding primer for the research of environmental DNA biodiversity in freshwater mussels. Environmental DNA technology can be used as an effective supplement to traditional methods in biodiversity monitoring. The reliability and detection rate can be increased by using multiple pairs of primers at the same time.