Abstract: In present study, Rhizopus chinensis 12 mixed fermentation with Bacillus sp. DU-106 were performed to improve the quality and flavor of Semen Sojae Praeparatum (SSP). The levels of physicochemical characteristics, bioactive compounds, free amino acids, and volatile components during the mixed fermentation process were monitored. Compared to SSP fermented only with R. chinensis 12, the levels of physicochemical characteristics were not obviously changed after mixed fermentation. However, thrombolytic enzyme activity and soy isoflavone content after mixed fermentation were significantly higher when compared to those of SSP fermented only with R. chinensis 12, and reached to 3927.84 IU/g and 3.91 mg/g, respectively. The amount of bitter amino acids and umami amino acids after mixed fermentation slightly lower than those of SSP fermented only with R. chinensis 12. The contents of flavor composition were considerably altered after mixed fermentation, of which, the pyrazine prevailed in SSP fermented only with R. chinensis 12, while, alkane predominated in SSP after mixed fermentation. Overall, the Rhizopus chinensis 12 mixed fermentation with Bacillus sp. DU-106 contributed to improve the thrombolytic activity, free amino acid content, and flavors of SSP.