In this study, a pair of specific primers and fluorogenic-labeled TaqMan-Minor Groove Binder(TaqMan-MGB) probe were designed and synthesized based on the conserved region of pirAVp gene, then a real-time PCR method was developed to detect AHPND-causing Vibrio parahaemolyticus(VpAHPND). The results showed that the method had a wide quantitative range from 1×101 to 1×109 copies per reaction, with a good linear relationship in its standard curve. The real-time PCR method had a high sensitivity with the detection limit as low as to 10 copies per reaction for the purified recombinant plasmids of pTOPO-T-pirAVp, and the entire detection could be completed within 1 h for a single sample and it also had a high specificity in detecting DNA of VpAHPND. The variation coefficients among cycle thresholds(Cts) of the repeatability test were 0.37%-0.47%. Comparing with the nested PCR recommended by OIE, the TaqMan-MGB probe real-time PCR had better positive detection rate of VpAHPND. In conclusion, the TaqMan-MGB probe real-time PCR assay developed in the study is fast, sensitive, specific, quantitative and of high reproducibility, making it a ideal method for detecting VpAHPND in shrimp and water samples.