In the design of recombinant bacterial vector vaccine, heterogeneous antigen is displayed on the outer membrane of the vector strain to evoke polyvalent immunological protection. Thus, the expression of heterogeneous antigen in cells and its display on the outer membrane are of great concern for vaccine preparation. In our previous work, a multivalent bacterial vector vaccine MVAV6203A-1 was constructed by displaying the protective antigen GAPDH from Aeromonas hydrophila on the surface of an attenuated Vibrio anguillarum MVAV6203. In this work, a new fermentation medium was designed by a four-step method to improve the cell growth and antigen display of V. anguillarum MVAV6203A-1. First, suitable carbon and nitrogen sources were selected by a component swapping method. Second, the initial concentrations of carbon and nitrogen sources were determined by orthogonal design. Then three main factors to significantly affect cell growth and antigen expression were screened by a Plackett-Burman design. Finally, the three main factors were meticulously optimized by response surface methodology. Based on this medium, a fed-batch fermentation process was established in a 5-L bioreactor, and the dry cell weight, the antigen expression in cells, and its display on outer membrane reached 5.98 g/L, 2.82 mg/g DCW, and 0.119 mg/g DCW, respectively.