Abstract: In order to enhance the flavor sensory attributes and quality stability of kombucha, utilization of specific strain based synthetic microbial communities (SMC) during the fermentation process was recognized as reliable strategy for kombucha production. In this work, tea fungus from three different origins of China were employed to isolate yeast and bacteria microbes suitable for SMC construction and kombucha fermentation. Based on the fermentation performance of each isolated strain, SMC was successfully reconstructed and employed for the kombucha fermentation process after optimization by response surface methodology (RSM) based on sensory scores of the fermented kombucha. A total of 12 microbial strains (8 yeast strains and 4 acetic acid bacteria strains) were isolated and identified from tea fungus of different regions, with two strains of Zygosaccharomyces bisporus (Y1-1 and Y3-2) and one strain of Gluconobacter oxydans (A3) were selected to reconstruct SMC for the production of raw Pu-erh tea kombucha. The RSM results showed that the optimal fermentation conditions were inoculation volume of 1%, sucrose concentration of 8%, tea concentration of 0.8%, and fermentation duration of 5 days, with the sensory score value of kombucha reached 8.38. The volatile flavor compounds of SMC fermented kombucha were determined by gas chromatography-mass spectrometry (GC-MS), the content of some key flavor compounds such as acetic acid and ethanol revealed to be 65.55±3.53 μg/L and 1.71±0.22 μg/L, respectively. Meanwhile, the total phenolic and flavonoid contents of the kombucha reached 1074.86 mg GAE/L and 1076.31 mg RE/L, respectively, with efficient in vitro antioxidant activity observed. Based on the selected autochthonous yeast and bacterial strains, the reconstructed SMC would provide scientific reference for improving the flavor sensory attributes and quality stabilization of fermented kombucha.