Abstract: In this study, headspace solid phase micro-extraction (HS-SPME) combined with gas chromatography-mass spectrometry (GC-MS) was adopted to separate and identify the volatile components of Kombucha by using polar column DB-WAX. Then, odor activity value (OAV) combined with stoichiometry was used to explore the volatile substance composition and characteristic differential substances of Kombucha in different regions. The results showed that there were significant differences between the volatile substances of Kombucha in different regions. A total of 56 kinds of volatile substances were detected, mainly alcohols, acids, esters and phenols. The OAV results showed that 32 kinds of substances contributed to the flavor for Kombucha (OAV≥1), the OAV of ethyl isovalerate of Kombucha in different regions was highest except for Urumqi and Weihai, it might play an important role in the flavor composition of Kombucha in different regions. Furthermore, with partial least squares discrimination analysis (PLS-DA), 17 variables with importance values greater than 1 as differential markers were identifed. By integrating the results of OAV and PLS-DA, a total of 14 characteristic compounds of Kombucha were screened out, namely 3-methyl-1-butanol, 2-ethyl-1-hexanol, phenethyl alcohol, nonanol, acetic acid, 3-methylbutyric acid, caprylic acid, ethyl acetate, isopentyl acetate, ethyl caprylate, ethyl phenylacetate, furan formaldehyde, 2,6-di-tert-butyl-p-cresol, and 4-ethylphenol. The research results will provide theoretical reference for the standardization, high-quality production and construction of characteristic brands of Kombucha.