In this study, we investigated the fish species composition of the Pearl River estuary using environmental DNA (eDNA) metabarcoding. DNA were extracted from water samples collected from 7 sites in Lingding bay of Pearl River estuary and partial sequences of the mitochondrial 12S were amplified. 26 777 effective sequences were obtained in total and the sequences were clustered into 35 OTUs (operational taxonomic units). A total of 35 fish species were identified from 9 orders, 22 families and 29 genera. Among them, 30 species were identified to the species level, and 5 species were only identified to the genus level. 9-31 species were detected separately in 7 sampling sites. Most of the species belongs to Perciformes, with 22 species in total. The vulnerable species (Taeniura meyeni) was detected at sites A and F. By comparing with two similar studies the Pearl River estuary, we found that the difference of the sampling area, sample collection strategy and the molecular marker adopted makes significant differences in the number and composition of the fish species detected. The results show that the environmental DNA metabarcoding technology is suitable for the investigation of fish species composition in Lingding Bay of the Pearl River estuary, which can be used as a supplementary method for the investigation and assessments of fish diversity in the estuary area.