During the fermentation process of natto, the decomposition of soy protein by Bacillus subtilis produces ammonia-nitrogen-like substances, resulting in a finished product with an ammonia-fishy odor, which affects consumer acceptance. In this study, Bifidobacterium bifidum, Lactobacillus lactis, and Bacillus subtilis were mixed for natto fermentation experiments, respectively, aiming to improve its flavor characteristics. The experimental results showed that fermentation with mixed strains significantly changed the volatile matter composition of natto. Among them, the electronic nose analysis showed that the content of nitrogen oxides and inorganic sulfides increased significantly in the fermented samples, while the ammonia nitrogen species decreased relatively. In addition, the amino acid analyzer test revealed that the total amount of free amino acids was significantly increased after fermentation of the mixed strains, while several flavor-improving amino acids also occupied a larger proportion. The experimental results indicated that the addition of Bifidobacterium and Lactobacillus could effectively regulate the metabolites during natto fermentation, reduce the ammonia-fishy odor, and improve the fermentation quality, which has potential application value.