Abstract: In order to study the effects of two enzyme-producing yeasts, Kluyveromyces marxianus (M19) and Pichia fermentans (M26) on the flavor of cheese, SPME-GC-MS and electronic nose detection were used to identify the volatile compounds in Cheddar cheese produced without (control) and with Kluyveromyces marxianus (M19) and Pichia fermentans (M26). The results showed that a total of 64 volatile compounds were detected across the three groups of cheese samples, including 10 acids, 16 alcohols, 19 esters, 6 aldehydes, 8 ketones, 2 lactones and 3 others. Principal component analysis (PCA) and cluster analysis of the detected volatile compound content and types revealed that the main volatile substances in the cheese group with M26 addition were acids, alcohols, esters, and ketones. The main volatile substances in the cheese group with M19 addition were esters, while the main volatile substances in the control group were alcohols. Comparative analysis of key flavor substances based on ROAV (Relative Odor Activity Value) during cheese ripening showed that the M19 group exhibited the highest number of volatile flavor compounds at 20 days of ripening, including acetic acid, isoamyl alcohol, n-amyl alcohol, ethanol, ethyl hexanoate, ethyl acetate, phenethyl acetate, ethyl decanoate, ethyl butyrate, and benzaldehyde. In contrast, the M26 group at 30 days of ripening primarily contained acetic acid, isoamyl alcohol, n-amyl alcohol, and ethyl hexanoate. Among these, ethyl hexanoate showed the highest ROAV in both treatment groups, indicating it as the most critical flavor compound in cheeses fermented with enzyme-producing yeasts, imparting distinct fruity and wine-like aromas. The odor of the M19 fermented cheese experimental group was strongest when the maturity period was 20 days, indicating that M19 fermented cheese could more efficiently promote cheese flavor formation, shorten the maturation period to 20 days, and help reduce production costs and improve efficiency. However, the flavor development ability of M26 fermented cheese was relatively weak, which might lead to a single flavor and a long maturation cycle of the product. These findings provide a valuable reference for the application of yeast in cheese production.