Objective： To establish a rapid method for the detection and identification of Vibrio parahaemolyticus by matrix assisted laser desorption/ ionization time of flight mass spectrometry （MALDI-TOF-MS）. Methods： In order to verify the stability and repeatability， different mediums， culture times and sample handing methods were optimized. The 461 isolates of Vibrio parahaemolyticus was used as a reference to evaluate the credibility of MALDI-TOF-MS method and analysis on mass spectrometric profiles. Results： Taking 2%NaCl-Tryptic Soy Agar Base as the culture medium， the mass spectrum with strong signal and good repeatability was acquired after 37 ℃， 24 h. The results showed that more characteristic peak of protein with smooth baseline， less noise and higher signal-to-noise ratio were obtained by ethanol/ formic acid treatment method. The 461 isolates of Vibrio parahaemolyticus was 97.6% consistency at the species level. Conclusion： MALDI-TOF-MS is a fast and accurate method for the identification of V. parahaemolyticus by MALDI-TOF-MS， and it can be used for routine detection because of its good stability and repeatability.