Abstract: This study aimed to develop Emmental cheese from yak milk enriched with conjugated linoleic acid (CLA). High-CLA-converting lactic acid bacteria were added to evaluate the changes in the quality and fat oxidation characteristics of the cheese. We simulated the ripening environment of Emmental cheese and screened the optimal strains to prepare direct-vat set (DVS) adjunct starters. The cheese added with DVS adjunct starters serves as the experimental group, and the cheese without such addition serves as the blank group. At 1, 25, 50, 75, and 100 days of cheese ripening, the microstructure, volatile compounds, fat degradation, and fatty acid composition and their variations of the experimental and blank groups were compared through sensory evaluation, scanning electron microscopy (SEM), headspace solid-phase microextraction-gas chromatography-mass spectrometry(HS-SPME-GC-MS), Soxhlet extraction, and gas chromatography-mass spectrometry (GC-MS). The results indicated that Lactobacillus fermentum 57 and Lactobacillus delbrueckii 60 were identified as the optimal strains. The microstructure of the experimental group was superior to that of the blank groups. Additionally, 2-nonanone and ethyl lactate were detected as unique flavor compounds of the high-CLA-converting lactic acid bacteria. With the extension of ripening time, the fat content of the three cheese groups first increased and then decreased, and was higher at 100 d than at the initial fermentation stage; both the acid degree value (ADV) and the thiobarbituric acid value (TBA) gradually increased with ripening time. Moreover, the TBA value of the experimental group was less than 0.5 mg/kg, ensuring no generation of off-flavors. The CLA conversion rates of the Lactobacillus fermentum S57 and Lactobacillus delbrueckii S60 groups of cheese reached up to 6.09% and 7.06% respectively, both of which were higher than that of the blank group. At 25, 50, and 100 days of ripening, significant differences in the health-promoting index were observed between the experimental and blank groups (P<0.05), with the experimental group showing a higher level of health promotion. In conclusion, the application of high-CLA-converting lactic acid bacteria in yak milk Emmental cheese can enhance its quality and improve fat oxidation, thereby demonstrating potential for development and application in academic and practical scenarios.