Acute hepatopancreatic necrosis disease (AHPND) is an important bacterial disease occurring early after stocking shrimp fry in shrimp ponds with the mortalities of 100 %. AHPND leads to significantly drop in production and brings out huge economic losses worldwide. Thus, rapid, accurate, and convenient on-site detection method is urgent need to monitor the outbreak and spreading of AHPND especially for equipment-poor areas. Application of traditional PCR-based methods is restricted due to the dependence on laboratory equipment and technicians. In this study, an improved isothermal recombinase polymerase amplification (RPA) combined lateral flow strip (LFS) assay was developed for AHPND detection by introducing a probe. The specific primers and probe were designed based on the PirAB gene, chemical modifications were labelled to improve the specificity, and mismatched bases were made to eliminate primer-dependent artifacts. In combination with crude DNA extraction by boiling for 10 min, the RPA-LFS assay could be finished within 25 min at 37-45°C and results were readable by naked eyes. The exclusivity was validated to be no cross-reactivity with 10 other common vibrio spp strains. The inclusivity was verified using 10 other VPAHPND strains isolated from infected shrimps. The limit of detection was 101 colony forming unit (CFU)/mL or 102 copies/μL and 100 CFU/10 g after 2 hours enrichment in spiked shrimp samples. The detection accuracy was evaluated in a total of 75 collected shrimp and seawater samples, which was proven to be consistent with AP4. The established RPA-LFS method provides a rapid, accurate, sensitive and equipment-free approach for on-site detection of AHPND and technical references for monitoring other pathogens in cultivation industry.