An acute hepatopancreatic necrosis disease (AHPND) causes serious losses to the global shrimp industry. The etiologic agent of AHPND is Vibrio spp. carrying a large plasmid which encodes a binary toxin, PirAB. Currently, AHPND is diagnosed by PCR based methods that detect the presences of both pirA and pirB genes. However, the bacterial strains containing the pirA and pirB genes do not always express the binary toxin, resulting in mis-estimation of the virulence of bacterial strains containing pirA and pirB genes. Thus, the immuno based assay (i.e. ELISA) is a promising approach to detect PirAVp and PirBVp. In the present study, a total of forty monoclonal antibodies clones (mAb) against PirAVp (20 mAbs) and PirBVp (20 mAbs) were screened by western blot analysis to select four mAb clones that show the strongest immunoreactivity in indirect ELISA (iELISA). The four selected mAbs (i.e. 1B9 and 5E9 against PirAVp; 7B7 and 7B9 against PirBVp) detected specifically Vibrio spp. causing AHPND. In addition, four selected mAbs were able to detect either PirAVp or PirBVp down to 0.008 ng/μl. A double blind assay using thirty AHPND-infected and six SPF shrimp Penaeus vannamei were analyzed by iELISA to determine the detection sensitivity of the assay. The results showed that iELISA was able to accurately detect 29 out of 30 AHPND infected shrimp. These finding indicated that iELISA is a reliable method to detect PirAVp and PirBVp toxins in infected shrimp and will be a useful tool in AHPND diagnosis and in studying the role of binary toxins in AHPND pathogenesis. •An iELISA using mAbs against Photorhabdus insect related toxins, PirAVP and PirBVP produced by Vibrio species was developed.•Four mAbs against PirAVP and PirBVP, that showed strong reactions in western blot analyses were selected for developing iELISA.•Using an iELISA PirAVP & PirBVP were detected in Penaeus vannamei shrimp infected with Vibrio parahaemolyticus causing AHPND.