Propidium monoazide (PMA) combined with molecular quantitative real-time PCR (qPCR) has been widely used for only detection of viable bacteria.In this study,an optimized viable multiplex qPCR combined with sarkosyl was developed for simultaneous quantification of thermostable direct hemolysin (tdh+),and thermostable-related hemolysin (trh+) genes of viable V.parahemolyticus in raw shrimp.The optimization of the treatment showed that the sarkosyl with lower concentration PMA and the dark incubate duration for 15 min with 15 min of light exposur could effectively inhibit 106 CFU/mL dead cells of bacteria.The high specificity was confirmed by using target and non-target strains.Furthermore,this assay has been successfully applied to detect and quantify oftdh+ and trh+ of viable V parahemolyticus in raw shrimps.In conclusion,the novel multiplex qPCR could be a rapid and effective diagnostic tool for the monitoring of viable V.parahemolyticus in food risk assessment.