Abstract: Interaction between microbial plays an important role in Baijiu brewing. Cyberlindnera fabianii and Clavispora lusitaniae are common non-Saccharomyces yeast in Baijiu brewing, but their interaction with mold is not clear. This study focused on sensory evaluation, content of volatile flavor substances, yeast biomass, enzyme activity and metabolites, and discussed the interaction between C. fabianii HY-11, C. lusitaniae HY-21 and Aspergillus oryzae M-08 in the brewing process. Results showed that, when two non-Saccharomyces yeasts were co-cultured with A. oryzae M-08, respectively, the biomass of C. fabianii HY-11 and C. lusitaniae HY-21 were 4.96 and 0.97-fold higher and alpha-amylases increased by 70.31 and 107.73-fold than that of pure fermentation using single yeast. These results indicated that the two yeasts were dependent on A. oryzae M-08. Compared with C. lusitaniae HY-21, the mixed fermentation of C. fabianii HY-11 and A. oryzae M-08 showed better flavor. After 6 days of fermentation, strong cantaloupe, wine and ester aroma were obtained. The total amount of volatile flavor substances was up to 1408.97 μg/g, which was 381.87-fold higher than that under single yeast fermentation condition. A. oryzae M-08 interacted well with C. fabianii HY-11 and C. lusitaniae HY-21. Compared with the single yeast fermentation system, the level of free amino acids in the mixed fermentation system increased by 105.02 and 3837.29 μg/g, and the esterase activity increased by 27.67 and 387.82 U/g, respectively. Therefore, the metabolism of volatile flavor substances was promoted. This study elucidates the relationship between two non-Saccharomyces yeasts and A. oryzae M-08 and their flavor characteristics, providing a theoretical basis for their rational application.