The taste and flavor of fermented vinegars are closely related to the fermentation microorganisms. In order to investigate the vertical distribution of bacterial community in the acetic acid fermentation stage of Zhenjiang aromatic vinegar, and to investigate the enrichment effect of different media on the culturable bacteria from Cupei. In this study, the bacterial diversity of Cupei and enriched culture samples were determined by high-throughput sequencing. The results showed that the number of OTUs in the surface, middle and bottom Cupei samples were 66, 76 and 111, respectively. The dominant bacterium in the surface layer was Acetobacter
, while the dominant bacterium in the middle and bottom layers was Lactobacillus
. The richness and diversity of bacterial flora became more abundant and diverse with the depth of fermentation. Meanwhile, high-throughput sequencing after enrichment showed that the oligotrophic medium enriched higher bacterial diversity than the nutrient-rich medium. This study revealed that there were differences in bacterial diversity in different depths of Cupei. And it also proposed an oligotrophic medium for bacterial isolation to make isolation of fermentation microorganisms from Cupei more efficient.