Barcode reference databases are an important foundation for DNA-based biomonitoring and an indispensable part of the current trending and promising environmental DNA (eDNA) technology. For effective DNA-based biomonitoring in the Pearl River Basin, the coverage or gap status of DNA barcodes of species is urgently needed to be captured. Here, we compiled a detailed historical record and species checklist of common fish and macroinvertebrates in the Pearl River. All available DNA sequences (e.g., the mitochondrial genome, Cytochrome c Oxidase subunit I (COI) gene, 12s rRNA gene, and 18s rRNA gene) matching the species names in the checklist of the common fish and macroinvertebrates were downloaded separately in the NCBI GenBank database, and analyzed the coverage of the DNA barcode records of species in the NCBI GenBank database. Our data showed that: (1) a total of 221 common fish species were recorded, including 2 classes, 18 orders, 51 families, and 137 genera, there are 2 endemic genera and 17 endangered rare species, and the common fish mainly included Cypriniformes, Perciformes, Siluriformes, the remaining 15 orders accounted for a combined 24.89%; A total of 6 classes, 14 orders, 53 families, and 105 species (or genera) of common macroinvertebrates in the Pearl River were retrieved, mainly Ephemeroptera, Trichopter, Plecoptera, the remaining 11 orders accounted for a combined 24.77%. (2) A total of 913 sequences of the mitochondrial genome and 962 sequences of 12s rRNA gene were retrieved, covering 81.45% and 57.92% of the total number of common fish species, respectively; 12.67% of the species had neither mitochondrial group sequences nor 12s rRNA gene sequences. The gap would rise to 52.94% if the barcode thresholds were set to contain at least five reference sequences per taxon. (3) A total of 65 sequences of the mitochondrial genome, 26988 sequences of COI gene and 175 sequences of 18s rRNA gene were retrieved, covering 29.52%, 68.57% and 37.14% of the total number of common macroinvertebrates species (or genera), respectively. 25.71% of the species (or genera) had no sequences included in the mitochondrial group, COI and 18s rRNA gene regions. The gap would rise to 41.90% if the barcode thresholds were set to contain at least five reference sequences per taxon. Overall, the collation of data in this study will provide importantly basic data support for DNA-based monitoring of fish and macrobenthos in the Pearl River Basin, and provide reference suggestions for improving the native DNA barcode database in the Pearl River.