Abstract: This study was conducted to investigate the differences of metabolic function and community composition in the phyllosphere of tobacco brown spot leaves at different maturity stages. The technologies of Biolog ECO metabolic phenotype and high-throughput sequencing were used to analyze three different maturity level tobacco leaves (mature, proper, immaturate) during brown spot occurring season. The results showed there were significant differences in the metabolic functions of tobacco phyllosphere microorganisms from three different maturity tobacco leaves. The highest metabolic activity was found in phyllosphere miroorganism from mature tobacco leaves, followed by the proper and immaturate tobacco leaves. L-Asparagine was the only carbon source that could be effectively metabolized in all three kinds leaf samples, while 2-Hydroxybenzoic Acid, L-Phenylalanine, α-Cyclodextrin, γ-Hydroxybutyric Acid and Phenylethylamine were all moderately or poorly metabolized in the three maturity leaves samples. The dominant phyla of fungi and bacterium of tobacco leaves at three maturity stages were Ascomycota and Proteobacteria, respectively. The dominant fungal genera were Alternaria, Cladosporium, Didymella and Symmetrospora, and the dominant bacterial genera were Pseudomonas and Sphingomonas. Among the three kinds of tobacco phyllosphere fungi, the relative abundance of Ascomycota and Basidiomycota in mature tobacco leaves was the highest, and the relative abundance of mature tobacco leaves was significantly higher than that of immature tobacco leaves. Among the three kinds of tobacco phyllosphere bacteria, the relative abundance of Proteobacteria in immature tobacco leaves was the highest and significantly higher than that in proper tobacco leaves. There were no significant differences in fungal community diversity and bacterial community diversity (Shannon, Simpson) for phyllosphere microorganisms between tobacco leaves at different maturity stages. However, the phyllosphere fungal community richness (ACE) of mature tobacco leaves was the highest, followed by that of proper and immaturate tobacco leaves, and the ACE of mature tobacco leaves was significantly higher than that of immature tobacco leaves.