Abstract: In order to develop a new type of high-quality honey tea wine, a mixed culture of Saccharomyces cerevisiae and non-Saccharomyces cerevisiae was employed for the fermentation of honey tea wine. The electronic nose technique in combination with headspace solid-phase microextraction-gas chromatography-mass spectrometry (headspace solid-phase micro extraction and gas chromatography mass spectrometry, HS-SPME-GC-MS) was adopted, and the basic physicochemical properties, organic acid, and polyphenol contents of the fermented honey tea wine were detected. The single-strain fermentation products of Saccharomyces cerevisiae (SF) and non-Saccharomyces cerevisiae (HF) were used as control groups for comparative analysis to comprehensively evaluate the effects of mixed-strain fermentation (MF) on the quality and flavor of honey tea wine. The results indicated that compared with SF and HF, MF increased the total phenol and total flavonoid contents of honey tea wine. The organic acid content in the post-fermentation samples increased, while the polyphenol content decreased. The response value of the electronic nose sensor for HF was low, and the majority of the sensor response values of MF were greater than those of SF. A total of 75 aroma components were identified by HS-SPME-GC-MS, and mixed-strain fermentation significantly increased the types and contents of volatile compounds in honey tea wine (P<0.05). A total of 10 differential compounds (VIP>1) were screened out by PLS-DA analysis, among which 6 were ester flavor substances. There were 12 key aroma components with OAV>1, and compared with HF and SF, those with higher contribution rates to the aroma of MF were phenylethanol with floral aroma, phenyl ethyl acetate with honey sweet aroma, pentyl formate and n-propyl acetate with fruit aroma. In conclusion, compared with single-strain fermentation, mixed-strain fermentation of honey tea wine has more advantages, with a richer and more intense overall flavor. This paper provides a theoretical basis for the application of non-Saccharomyces cerevisiae in alcoholic beverages and the deep processing and utilization of tea by-products.