Abstract: Mixed fermentation has exhibited great potential in improving flavor quality of wine and fruit wine. To reveal the competitive utilization of amino acids by different yeasts and their effects on key aroma production in mixed fermentation, this study used Saccharomyces cerevisiae and Hanseniaspora osmophila as research subjects, prepared simulated grape juice as the fermentation medium model. Single and mixed fermentations were conducted to assess fermentation activity, cell growth, amino acid consumption, and aroma production. Mixed fermentations included two modalities (simultaneous and sequential inoculation), each yeast was inoculated at a cell concentration of 2×10⁶ CFU/mL, and the inoculation ratio was 1:1. In sequential fermentation, H. osmophila was inoculated firstly, and S. cerevisiae was inoculated after 48 h. Results showed that both S. cerevisiae and H. osmophila preferentially consumed most amino acids, while the former exhibited higher uptake rates. In sequential fermentation, H. osmophila consumed key amino acids (alanine, aspartic acid, glutamic acid, threonine and serine) related to cell growth, which significantly reduced the maximum biomass of S. cerevisiae (−33%, P<0.05) subsequently inoculated, and induced fermentation delays. S. cerevisiae dominated amino acid utilization in simultaneous fermentation, leading to a similar production of aroma compounds compared to single S. cerevisiae fermentation. Aroma production in sequential fermentation was conditioned by amino acid competition between two yeasts. In sequential fermentation, early utilization of amino acids by H. osmophila induced the production of higher alcohol acetates (particularly 2-phenylethyl acetate, whose OAV was increased by 15-fold), and high productivity of fatty acid ethyl esters in S. cerevisiae was also retained. This study characterized the competitive utilization of amino acids and key aroma production in mixed fermentation, providing a scientific basis for improving fermentation stability and aroma-enhancing efficiency of mixed fermentation through exogenous supplementation of key amino acids.