Abstract: To establish an efficient collection system for tobacco leaf surface microorganisms, high-secreting tobacco T.I.1068 was used as the research material. Solutions of Tween-80 at concentrations(volume fraction)of 0, 2%, 8%, 10%, 15%, 20%, and 30% were used to elute and collect leaf surface microorganisms. The effects of Tween-80 on the elution of leaf surface microorganisms from high-secreting tobacco were studied by plate culture and amplicon sequencing. The results are as follows: 1)With the increase of Tween-80 concentration, the number of cultivable microorganisms on the plate first increased and then decreased. The bacteria colony density was the highest under the treatment of 15% Tween-80, and that of fungi was the highest under the treatment of 10% Tween-80. 2)With increasing Tween-80 concentration, the total DNA concentration of the foliar microorganisms showed an increasing trend and reached a plateau at 20% or above. 3) 16S rDNA amplicon sequencing analysis showed that the richness and evenness of bacterial species obtained by 20% Tween-80 elution were higher than those of aqueous solutions, but the structure of the community of bacteria changed, and the abundance of bacterial families such as Xanthomonadaceae, Xanthomonadaceae, Sphingomonadaceae, Rhizobiaceae and Pseudomonadaceae increased significantly. 4)Amplicon sequencing and ITS rDNA analysis showed that the richness of fungal species obtained by 20% Tween-80 elution was lower than that of the aqueous solution, but the structure of the fungal community changed, and the abundance of Erysiphaceae and Phaffomycetaceae, the fungal families, increased significantly. In conclusion, Tween-80 could effectively improve the efficiency of microbial elution and the concentration of total microbial DNA, and had a positive effect on microbial culture and community structure and diversity analysis.