Abstract: To explore new ways for observing and protecting fish species diversity in the middle and lower reaches of the Pearl River, we used eDNA technology to detect fish species diversity in that river section. Six sampling localities, namely Guiping, Tengxian, Fengkai, Deqing, Zhaoqing and Jiujiang, were set up in February 2023. The fish diversity was detected by eDNA metabarcoding analysis that includes water collection, water filtration, eDNA extraction, genetic marker amplification, sequencing and bioinformatic analyses. The results show that 30 fish species had been detected in 6 sampling sites, belonging to 4 orders, 10 families and 27 genera, in which 26 were native species and 4 were non-native species. Compared with traditional survey reports, two new species (Botia pulchra and Oceochromis zillii) were detected. Rhinogobius giurinus, Pelteobagrus vachellii, Hypophthalmichthys molitrix, O. nilotica, O. zillii, Rasbora steineri and Cyprinus carpio were found at each sampling site, indicating that these species are the dominant species in the survey area. According to Shannon index and Simpson index, the fish diversity was highest in Jiujiang and Guiping, while lowest in Tengxian. As a new detection method, eDNA technology can efficiently investigate the fish biodiversity and distribution in the middle and lower reaches of the Pearl River. To sum up, eDNA technology can be combined with traditional fish resources monitoring methods to provide more comprehensive data information on fish species diversity.