Abstract: In order to investigate the content of nutrients (free amino acids , fatty acids) and flavor substances in koumiss fermented with Xinjiang-derived superior yeast strains, as well as the changes in substances before and after fermentation, and the differences in nutrients and flavors compared with that of naturally fermented koumiss, ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS), gas chromatography-mass spectrometry (GC-MS), and comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC×GC-TOFMS) were employed to detect the contents of free amino acids, fatty acids, and volatile flavor substances in fresh mare's milk, naturally fermented koumiss (control group), and koumiss fermented with superior yeast strains (experimental group) . The results showed that, compared with fresh mare's milk, the content of free amino acids in the experimental group significantly increased, while the relative content of polyunsaturated fatty acids significantly increased, the number of volatile flavor substances increased, wherein the relative contents of esters, alcohols, carboxylic acids, and heterocyclic compounds were elevated. Compared with the control group, the essential amino acid content in the experimental group significantly decreased, while the contents of histidine (1297.68 mg/L), ornithine (545.38 mg/L), and γ-aminobutyric acid (1269.46 mg/L, GABA) significantly increased. The relative content of saturated fatty acids significantly decreased, while the relative content of polyunsaturated fatty acids (PUFAs) significantly increased, wherein the relative content of α-linolenic acid (19.72%) was significantly enhanced, but the relative content of linoleic acid (9.32%) significantly decreased. The total number of flavor substances in experimental group was reduced, and the key flavor substances were identified as 2,3-butanedione, 2-undecanone, heptanal, and 2-pentylfuran. Although the diversity of flavor substances was relatively lower, the overall flavor profile was more harmonious, characterized by buttery, cheesy, grassy, and fatty notes. In summary, the mixed yeast fermentation intervention effectively boosts the nutritional quality and optimizes the flavor balance of koumiss.