Objective To establish a rapid identification method of Vibrio alginolyticus using matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Methods The effect of different sample pretreat method (direct smear method and formic acid extraction method), laser intensity and times of subculture on the quality of spectrum was investigate using the reference strain CICC 10889. The impact of database on identification result was evaluate by comparing the identification result (species and Logscore) of 36 isolated strains before and after adding the main spectra product (MSP) of CICC 10889 to the database. Results The formic acid extraction method was better than the direct smear method. Spectra of ethanol/formic acid treatment got more characteristic peak of protein with smooth baseline,less noise and higher signal-noise ratio. Laser intensity could affect spectra. High quality spectra could be obtained when the laser intensity was appropriate. Database was the most important impact factor. The Logscore of identification result was improved greatly by adding the in-house database, score of isolated strains were all above 2.300. Times of subculture had no effect on spectra. Conclusion MALDI-TOF MS could accurately identify Vibrio alginolyticus to the species level. The established MALDI-TOF MS method was accurate and constant and could be used to identify Vibrio alginolyticus rapidly.