Abstract: Based on the use of defined medium with selective pressure and the feeding of soy peptone, a two-stage fermentation strategy was developed to produce HBsAg by the recombinant Saccharomyces cereviciae. As a result, both the final production of HBsAg and HBsAg yield per cell increased by 25% and 68% respectively. Through the optimization of the defined medium, the two-stage fermentation became (economical) and potential in practical application. By the measurement of plasmid-bearing cells, it was shown experimentally that the increase of antibody expression was due to the improvement of plasmid stability.