Acute hepatopancreatic necrosis disease (AHPND) is currently the most affected disease of shrimp farms worlwide As rapid and specific detection methods are essential to improve disease management, a lateral flow immunoassay (LFIA) was designed for the detection of PirAvp and PirBvp toxins in shrimp. In this study, polyclonal antibody based lateral flow immunoassay (LFIA) strip has been developed. The recombinant PirAvp and PirBvp toxin‐like proteins of Vibrio parahaemolyticus (VPAHPND) was used to immunize rabbits with the highest antibody titre after the third booster. This polyclonal rabbit antiserum was purified and used to develop a LFIA strip for the detection of AHPND pathogen. This test strip could detect PirAvp and PirBvp toxins up to 125 ng/mL by naked eye within 15 min. No cross-reactivity was observed with VPnon-AHPND. Furthermore, the sensitivity and specificity of LFIA were 94.0% and 98.0%, respectively. This LFIA strip could be used as a handy tool by the farmers and technicians in situ.