Abstract: In order to establish a DNA barcode background database of fish in the Pearl River Estuary and provide information basis for fish species identification and diversity research, we had collected 251 samples from the Pearl River Estuary from 2020 to 2021, and determined 652 bp fragments of mitochondrial cytochrome oxidase subunit I (COI) of 219 individuals and 163−185 bp fragments of mitochondrial 12s rDNA of 247 individuals of 99 species, 10 families, 41 genera, 6 orders. Besides, we downloaded 165 COI sequences and 128 12S rDNA sequences from GenBank database, and had obtained 384 COI sequences and 375 12S rDNA sequences of 172 species of fish. The results show that the average intraspecific genetic distance of COI sequence was 0.20%, and the average interspecific genetic distance was 25.54%. The average intraspecific genetic distance of 12S rDNA sequence was 0.12%, and the average interspecific genetic distance was 34.39%. The DNA barcode of COI gene could form an obvious barcode gap, but the DNA barcode based on 12s rDNA gene could not, and it was difficult to distinguish the 11 species (6.4% of the total species). The establishment of the DNA barcode database of fish in the Pearl River Estuary is conductive to the environmental DNA analysis of the fish ecosystem in that area, providing reliable technical support for the protection of fish biodiversity and the monitoring of population dynamics in the Pearl River Estuary.