Objective To establish and evaluate a duplex polymerase chain reaction(DPCR)system of species-specific tlh gene and typical virulence gene (tdh1_368) of Vibrio parahaemolyticus (VP). Methods The DPCR system was evaluated by single blind method for specificity and sensitivity, and the field methodological evaluation was carried out by artificially contaminated samples, diarrhea fecal samples and aquatic products. Results The developed method had high specificity (100%) and high sensitivity (≥1.5 ng/μl). No significant difference in the VP positive rate of diarrhea samples and aquatic products detected by this method and culture method. This method confirmed that the VP epidemic strain within diarrhea samples was 93.5% (29/31)；DPCR could report VP epidemic clones within 24 hours with diarrhea specimens and 4-8 hours after enrichment of artificially contaminated samples. Conclusion DPCR combined with routine bacterial enrichment method could shorten the reporting time of monitoring and processing time of emergency samples, DPCR could help improve the prediction, early warning and traceability of VP epidemic clones, and improve the efficiency of public health disposal and control.