We designed a pair of specific primers and a TaqMan fluorescent probe targeting the toxR gene of Vibrio parahaemolyticus (VP). After optimizing the conditions, the specialty, sensitivity and reproducibility of the detection method were evaluated. Results: (1) the developed real-time PCR assay protocol detected only VP and was not affected by other normal food pathogens such as Staphylococcus aureus, Salmonela, Listeria monocytogenes. (2) the limit of detection was 25 copies of toxR gene in the detected samples, and the sensitivity of pure cultures and simulated food samples was 21cfu/ml and 210cfu /g. (3) the developed protocol of real-time PCR assay had a high reproducibility, and the sample’s variation was 0.9% and 1.3% within the same sample and between tests. (4) the standard curve had a good linearity when the gene quantity was between 2.5×101 and 2.5×106 copies. The developed detection assay targeting the toxR gene can quantitatively detect VP in only 3 hours, and thus is an efficacious method for the detection of Vibrio parahaemolyticus.