To develop a multiplex real-time quantitative PCR assay for detection Vibrio parahaemolyticus (Vp), Salmonella (Sal) and Listeria monocytogenes (Lmo) in seafood.Methods The primers and TaqMan probes were designed with tlh, Ompc and hly gene as target sequence, corresponding V.parahaemolyticus, Salmonella and L.monocytogenesfor the multiplex real-time PCR assay, and its specificity and sensitivity were then tested. 150 samples collected from Zhoushan were screened for V.parahaemolyticus, Salmonella and L.monocytogenes using this method compared with national standard. Results Only V.parahaemolyticus,Salmonella and L.monocytogenesstrains exhibited typical signal curves while other bacteria often concurred with them in seafoods were not amplified, and did not interfere with the detection, indicating the specificity of this assay. The detection limit of this method was 72 cfu/ml (Vp), 40 cfu/ml (Sal) and 80 cfu/ml (Lmo). In the 150 samples collected from Zhoushan, 32 samples were positive for Vp, 11 positive for Sal, 5 positive for Lmo which was consistent with national standard.Conclusion This method is specific, sensitive, rapid and easy for detection of V.parahaemolyticus, Salmonella and L.monocytogenes in seafoods.