Vibrio parahaemolyticus, is a common and important pathogen that causes human gastroenteritis worldwide. A rapid, sensitive and specific assay is urgently required for detection and differentiation of Vibrio parahaemolyticus strains. We designed three sets of primers and probes using the groEL, and two virulence genes (tdh and trh) from V. parahaemolyticus, and developed a multiplex real-time PCR protocol. The sensitivity and specificity of the multiplex assay was evaluated by V.parahaemolyticus strain from environment and clinical specimens. The multiplex PCR response system and annealing temperature were optimized. The detection limits of the multiplex real-time PCR were 104 CFU/mL and 105 CFU/ mL (g)in pure cultures and spiked oysters, respectively. The multiplex real-time PCR specifically detected and differentiated V. parahaemolyticus from 35 Vibrio strains and 11 other bacterial strains. Moreover, this method can detect and distinguish virulent from non-virulent strains, with no cross-reactivity observed in the bacteria tested. This newly established multiplex real-time PCR assay offers rapid, specific and reliable detection for total and pathogenic V. parahaemolyticus strains, which is very useful for rapid detection of total and pathogenic V. parahaemolyticus during outbreaks and sporadic cases caused by V. parahaemolyticus infection.