Abstract: In order to collect microbiota efficiently from tobacco leaves during aging process and obtain the most suitable separating agent and conditions. The K326 toabacco with WDC3F grade from Yuxi area were used as the experimental material in this study. The bacterial floras of aging tobacco leaves were collected by using four separation solutions. Meanwhile, the collected bacterial flora was cultured and amplified. The bacterial flora diversity was also evaluated before and after the culture using culturable and non-culturable analysis. The results showed that an effective method for collecting microbiota of aging tobacco leaves with 1‰ SDS as a helper in separation solution was found that the collection rate was increased by 489.58% compared with the traditional method., The tobacco leaf infusion medium supplemented with tobacco leaf extract could not only increase the total number of bacterial flora by 100 folds, but also preserve the flora diversity under the culture condition of 15℃. The results by non-culturable analysis showed that the structures of bacterial flora in amplified sample were changed, but there was no significant differences in the bacterial community metabolic function, which indicated that the amplified bacterial flora kept the same physiological functions as original bacterial flora. So, this study established an efficient collection and amplification method for the bacterial flora from aging tobacco leaves, which provided a basis for the development of aged tobacco leaf microorganism resources.