Vibrio harveyi is one of the most serious marine pathogen that can infect a number of aquaculture species. It has caused severe losses to aquaculture industries worldwide. Attempts to control the infection are hampered by lack of effective vaccines and rapid diagnostic kits, the formulation of which could be facilitated by the identification of immunogenic proteins. In this study, an immunoproteomebased approach was developed to identify candidate antigens of Vibrio harveyi for vaccine development. A 2DE map has been constructed for Vibrio harveyi, in the pI range of 4.0 to 7.0. Strain HY99 was grown in TSA medium for 18 hours at 28 ℃. Total soluble proteins were extracted using lysis buffer and purified with a 2D cleanup kit. Protein concentrations were determined by 2D Quant Kit, and the proteins were separated by 2DE under immobilized pH gradients (IPG). The 2DE map was obtained from 3 gels run with 7 cm immobilized pH gradient strips and 12.5% SDS-PAGE gels. The electrophoregrams were obtained by coomassie brilliant blue staining. 2DE gels were scanned with Image Master 2D Platinum analyzed by 300 dpi 2DE image analysis revealed (429±18) protein spots. Then Vibrio harveyi HY99 antisera was analyzed for reactivity by Westernblotting against Vibrio harveyi total soluble proteins separated by 2DE. The 3 maps analyzed revealed 45 pair protein spots by ImageMaster 2D Platinum. 15 spots are nonspecificimmunoreactive proteins of Vibrio harveyi, and 30 spots are specificimmunoreactive proteins of Vibrio harveyi. These 30 spots were chosen for mass spectrometry identification ,and 29 spots were successfully matched with the proteins of NCBInr database（http://www.matrixscience.com）. Two isoforms of formate acetyltransferase were proposed. The 30 spots from the 2DE map corresponded to 28 proteins. None of these identified proteins have previously been reported as immunogenic in Vibrio harveyi. 6 proteins are known from other bacterial immunoproteomic analyses. They may be considered to be crossreactive antigens from other bacterial infections. OmpN were identified a number of times during the immunoproteome analysis of other bacteria, such as Shigella flexneri, Pasteurella multocida, Escherichia coli. OmpW is one of the major outer membrane proteins of Vibrio alginolyticus, and it is an immunoprotein in the report. OmpU is an important virulence factor involved in the adherence of Vibrio vulnificus to the host cells. alanine dehydrogenase, Elongation factor Ts (EFTs), cysteine synthase were recognized by anti-sera of Staphylococcus epidermidis. To the best of our knowledge, there are no reports about the immunogenicity of the other remaining 18 identified proteins. Their role in immunoreaction is not fully understood. It is suggested that this study may be valuable for the immunoproteomics research on Vibrio harveyi. These immunoreactive proteins could be novel candidates for vaccine development. Future studies will evaluate the protection of the 28 proteins by a nasal immunization and challenge.