To investigate the possibility of TolB from Vibrio alginolyticus HY9901 as a candidate antigen for vaccine production, the tolB gene was amplified by using a pair of primer according to the published tolB gene sequence (JQ846501), and then followed by digestion, connection; the prokaryotic expression vector pET-TolB of tolB gene was constructed. Then the IPTG concentration, induction time, temperature, elution concentration of the pET-TolB was optimized to obtain a relatively large amount of the target protein. The result showed that, the TolB protein was successfully expressed after being inducted with IPTG and SDS-PAGE, Western-blot analysis. The molecular weight of TolB protein was in line with expectation, and the expressed protein existed in the form of inclusion body. The optimization condition of inducible expression for TolB protein in Escherichia coli was optimized at 37℃ for 4 hours, with 0.2 mmol/L IPTG. The best elution concentration of imidazole was 400 mmol/L. These results laid a foundation for further studies on the protein immunogenecity and vaccine preparation.