Abstract: To clarify the substance metabolism and the differences in metabolome of upper leaves of flue-cured tobacco during ripening, the metabolites of upper leaves 20, 30, 40, 50, 60 days after topping were analyzed by GC-MS non-targeted metabonomics technique combined with determination of hormones and pigments. The results showed that the ripening process of upper leaves could be roughly divided into three stages, 20-30 days, 40-50 days and 60 days after topping. With the increase of leaf maturity, the contents (mass fractions) of chlorophyll and carotenoid decreased gradually, which were the most obvious on the 40th day after topping. The contents of free amino acids and total nitrogen presented a downward trend, and the change of total alkaloids and carbohydrates contents were upward generally. The contents of growth-promoting hormones in tobacco leaves decreased significantly, while those of senescence-promoting hormones increased significantly on the 40th day after topping. The 40th day after topping might be the key point when the physiological function of upper leaves changed from thriving to senescence. Through orthogonal partial least squares discriminant analysis (OPLS-DA), 38 differential metabolites were screened out, in which amino acids, carbohydrates and fatty acids, including 4-aminobutyric acid, fructose, allose, palmitic acid, linolenic acid, glyceric acid, malic acid, scopoletin, quinic acid, might be the key metabolites of upper leaves in the ripening process. The differential metabolites were significantly enriched (p < 0.05) in the pathways of glucose metabolism, amino acid metabolism and fatty acid metabolism; among them, the pathways of glyoxylate and dicarboxylate metabolism, biosynthesis of unsaturated fatty acids, TCA cycle, alanine, aspartate and glutamate metabolism played important regulating roles in tobacco leaf ripening.