Atractosteus spatulais （alligator gar）， as a key regulated invasive species in China's Catalogue of Key Managed Invasive Alien Species， has posed a severe threat to local aquatic biodiversity. To meet the demand of rapid and accurate detection during the early stages of alligator gar invasion， this study developed a specific， highly sensitive and rapid detection system based on environmental DNA （eDNA） technology. A set of alligator gar -specific recombinase polymerase amplification （RPA） primers and probes was designed by mitochondrial genome alignment analysis. The validation results indicate： （1） The primer-probe set stably and efficiently amplified target DNA from tissue samples， aquaculture water samples， and aquaculture water samples from the Pearl River Fisheries Research Institute Chinese Academy of Fishery Sciences， Guangzhou， Guangdong， China. （2） Specificity validation demonstrated the primer-probe set produced positive signals exclusively for A. spatula is with no cross-reactivity to 11 non-target fish species， including Lepisosteus oculatus. （3） Sensitivity validation demonstrated the detection limit reached 15.88 copies/μL DNA for the primer-probe set. （4） RPA amplification can be completed within 15 min at 38 ℃ and the lines can be read within 5 min combined with lateral flow （LF） dipsticks. This study firstly established a rapid detection system for A. spatulais using RPA-LF based on eDNA， providing technical support for early warning and scientific management of invasive species in aquatic ecosystems.