Abstract: Fish biodiversity in the Pearl River Estuary is declining due to excessive fishing intensity and marine development. Fish biodiversity assessment is an important part of ecosystem protection and management. However, traditional fish biodiversity assessment tools often have limitations, and the new environmental DNA (eDNA) matebarcoding is a potential biodiversity assessment tool, which can monitor the whole ecosystem quickly and comprehensively. In this study, fish biodiversity was studied at 10 sites in the Pearl River Estuary in March 2020 through a standardized process of eDNA metabarcoding analysis, including water collection, water filtration, eDNA extraction, genetic marker amplification, sequencing and bioinformatic analyses. In addition, bottom trawling method was used for sampling at 9 stations at the same time. We compared the detection results of the two methods. 175 species of fish were detected by eDNA matebarcoding, and 47 species were collected by bottom trawling. Combined with the two methods, 179 species of fish were detected, belonging to 15 orders, 63 families and 128 genera. The two methods jointly identified 43 species of fish, accounting for 24.02% of the total detected species, the survey based on bottom trawling failed to collect most species detected based on eDNA matebarcoding. According to shannon index and Simpson index, the Alpha diversity of fish community detected by eDNA matebarcoding in the Pearl River Estuary was significantly higher than that of bottom trawling (P<0.05). The PCoA results of the two methods provided insight on the spatial structure of fish community in Pearl River Estuary, in which the analysis based on eDNA metabarcoding showed that there is more spatial overlap. Both methods based on redundancy analysis (RDA) showed that dissolved oxygen and salinity are the main environmental factors affecting fish community structure. This study shows that the eDNA metabarcoding is an environmentally friendly and reliable assessment method, and it can be better understood in the estuary fish diversity by carrying it into the existing survey.