Based on Vibrio parahaemolyticus real-time fluorescent quantitative PCR method, rapid detection method of Vibrio parahaemolyticus was developed.Methods According to the Vibrio parahaemolyticus gene sequences published in GenBank, specific target genes were selected for primers and probes design, and the reaction system was optimized. An internal amplification control (IAC) was added to the raction system. This IAC was detected by TaqMan probes labeled with different fluorophore. The samples were artificially contaminated in 5-50 cfu/25 g, and were used to evaluate the performance of the reaction system. Results The assay could be used reliably for detection of Vibrio parahaemolyticus with the sensitivity of 1 pg/μl. For the 10-fold dilutions bacteria DNA extracted by cooking water, the lowest detection limit was 4×10² cfu/ml; and for the plasmid with gyrB, the lowest detection limit could reach 100 copies/μl. The standard curves of gyrB and gyrB-IAC were established, which the quantification was linear between Ct and template copy number (r²=0.999). When the initial sample amount of artificially contaminated bacteria was 7 cfu/25 g seafood, the Vibrio parahaemolyticus could be detected after 6 hours culture.Conclusion The gyrB-IAC fluorescence quantitative PCR assay was developed. It could not only be applied for detection of Vibrio parahaemolyticus in food, but also monitor the PCR reaction system to prevent ‘false negatives'. Therefore, the gyrB-IAC fluorescence quantitative PCR assay further ensures the reliability of the results and is helpful to standardize quantitative PCR method for Vibrio parahaemolyticus in seafood.