Pistia stratiotes L. （water lettuce）， as a highly detrimental invasive plant species in China's freshwater ecosystems， has posed significant threats to aquatic ecological security through its rapid proliferation. To enable early monitoring and control of this species， this study developed a highly sensitive and specific detection system based on environmental DNA （eDNA） technology. By integrating k-mer indexing strategies with chloroplast genome alignment analysis， we designed four sets of species-specific multiplex PCR primers （PS_7， PS_8， PS_9， PS_10）. Experimental validation demonstrated that: （1） All individual primers and their combinations effectively amplified target DNA from tissue samples， aquaculture water， and natural water samples； （2） The detection limits reached 0.69×10^-2 ng/μL for individual primers PS_7 and PS_10， 0.34×10^-1 ng/μL for PS_8， and 0.17 ng/μL for PS_9. The multiplex PCR system achieved a detection limit of 1.1×10^-5 ng/μL， representing approximately a 625-fold improvement in sensitivity compared to the best single primer （P<0.01）； （3） Specificity tests confirmed no cross-reactivity with closely related species such as Spirodela polyrhiza. The entire detection process can be completed within 3 hours. This established detection system provides reliable technical support for early warning and monitoring of Pistia stratiotes invasions， while serving as a practical model for developing rapid eDNA-based detection methods for aquatic invasive plants.