Optimal control of microbial fermentation process is an important factor to improve yield stability and conversion rate. Traditional technology has some limitations in dealing with fuzziness and uncertainty in microbial fermentation. In this study, fuzzy ant colony algorithm is used to optimize the yield and conversion rate in microbial fermentation. First, fuzzy rules are established to describe the relationship between substrate concentration, temperature, pH value, yield, and conversion rate. Second, the key parameters are optimally controlled by fuzzy rules. Finally, the effectiveness of fuzzy ant colony algorithm is evaluated through numerical simulation experiments and actual microbial fermentation experiments. The experimental results show that the control optimization of microbial fermentation process proposed in this paper can obviously improve the yield and conversion rate.