Abstract: The Pearl River Basin is one of the most biodiversity-rich regions for aquatic organisms in China. In recent years, however, biological invasions by alien species have posed a severe challenge to its biodiversity, and early warning is crucial for their prevention and control. This study aimed to monitor the current distribution of alien fish species and assess their invasion risk in the Pearl River Basin using environmental DNA (eDNA) metabarcoding. A total of 19 sampling sites were established across the Dongjiang River, Xijiang River, and Beijiang River, and surveys were conducted in January and July 2022. Alien fish diversity was analyzed based on eDNA metabarcoding, and invasion risks were assessed under current and future climate conditions using the Aquatic Species Invasiveness Screening Kit (AS-ISK). In total, 16 alien fish species were detected, with 14, 13, and 15 species recorded in the Dongjiang River, Xijiang River, and Beijiang River, respectively. Compared with historical records, the number of alien fish species in each river section has increased. Risk assessment indicated that under current climate conditions, 10 alien fish species posed high invasion risks and 6 posed medium risks. Under climate change scenarios, Oreochromis aureus shifted from high to medium risk, while the risk levels of the other species remained unchanged. These results suggest that the Pearl River Basin is under considerable invasion pressure from alien aquatic species. Alien fish generally possess broader tolerance ranges, stronger adaptability, and higher reproductive capacity than native species, highlighting the urgent need for strengthened scientific prevention and control. This study provides a novel approach for monitoring and early warning of alien fish species and offers scientific references for the protection and management of aquatic biological resources in the Pearl River Basin.