OmpT, a major outer membrane protein of Escherichia coli, had a good prospect in vaccine development for its better immune detection. The expression strain of OmpT was obtained employing molecular clone technology and purified using gel slices approach, and the polyclonal antibody was prepared from immunized mice. The antibody titer tested by ELISA approach was 1∶1 600, and the antiserum tested by Western Blotting method had good specificity. From the results of orthogonal experiment, the optimal condition for inducing expression of OmpT protein were 1.0 OD600 nm value of strain, 0.3 mmol/L final concentration of IPTG, 8 h of inducing time and maintaining temperature in 32 ℃; The optimal cultural condition for glucose concentration, rotation rate, and medium volume were 0%, 230 r/min and 50 mL, respectively. These work provided a ground for industrial fermentation, protein function and vaccine development of OmpT.