Objective To obtain the best strain combination for fermenting traditional kombucha by screening the bacterial strains.Methods The strains isolated from traditional kombucha are evaluated in terms of the production of acids and alcohols. Three strains of acetic acid bacteria and four strains of yeast screened out are combined to form 12 fermentation groups for the production of kombucha, which are analyzed by gas chromatography-mass spectrometry (GC-MS) combined with the physicochemical and microbiological assays and sensory evaluation of the products.Results The acidity of the fermentation groups M6 (Komagataeibacter saccharivorans and Saccharomyces cerevisiae) and M12 (Acetobacter and S. cerevisiae) at the fermentation endpoint is 0.537 g/100 g and 0.495 g/100 g, respectively, higher than that of most fermentation groups. The microbial counts of the two groups reach the average level of all the fermentation groups. The two groups show significant differences in content of tea polyphenols, caffeine, and ethanol compared with some fermentation groups (P<0.05). GC-MS results reveal that the characteristic flavor compound in M6 and M12 groups is ethyl acetate, with the mass fractions of 20.12% and 19.08%, respectively. The results above combined with the sensory evaluation scores suggest that the M6 and M12 groups are superior to the other fermentation groups in terms of flavor, color, and sensory quality.Conclusion The M6 and M12 fermentation groups can be used as effective strain combinations for the production of traditional kombucha, improving the physicochemical indexes, flavor compounds, and sensory quality of traditional kombucha.