Abstract: Commercially available Kefir fermented milk does not have the taste of traditional kefir milk like champagne. In order to develop a pure Kefir fermented milk and prepare a new type of kefir starter culture, an excellent yeast strain is required to be selected with a moderate production of bubbles and resistance to high acidity and high temperature. In addition, such strain should have the unique flavor of Kefir milk that ordinary single lactic acid bacteria fermented yogurt does not have. Four aroma-producing yeast strains were isolated and purified from melon koji (a natural starter used to ferment sourdough steamed bread) by YPD medium, which were numbered TN₁, N2, Z2 and HX, respectively, to conduct subsequent experiments. The preliminary screening of four isolated strains resistant to acid, alcohol, and high termperature was carried out. Through solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS), the aroma components in the fermented milk fermented by the two yeasts obtained from the previous experiment were used as evaluation indexes, and the aroma components were rescreened. A strain with the best aroma content, type and flavor was selected. The final strain was identified by molecular biology. The results showed that after comprehensive evaluation of various indicators, the growth, tolerance, and fermentation performance of TN₁ and N2 in the four yeast strains were better. In comparison with N2 strain, TN₁ strain produced the most kinds of aroma substances, the content of typical flavor substances of which was moderate. The aroma of TN₁ strain had wheat alcohol aroma, rose aroma and cream aroma, which was better than the other strains. Through PCA analysis, the data were reduced in dimension, and the difference of key flavor substances between TN₁ and N2 was more intuitive and convenient. Therefore, TN₁ was more suitable for the subsequent development of Kefir yogurt. In combination with the molecular biological analysis results of TN₁ strain, the homology between the strain and NRRL Y-12632 strain was 100%, hence TN₁ was identified as Saccharomyces cerevisiae. This study provides a foundation for the development of Kefir fermented milk starter.