Two hundred and eleven hydrophilic metabolites and 74 lipophilic metabolites from tobacco leaves of flue-cured tobacco variety G80 at different maturity stages (on the day of topping and on the 10th, 20th and 30th days of topping) were identified using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). A total of 94 differential metabolites were screened out, including nine major groups: amino acids, sugars and glycosides, lipids, amines, alkaloids, phenylalanine and its derivatives, terpenes, nucleotides, and others. The key metabolites in tobacco leaves during the maturation process of flue-cured tobacco variety G80 were found to be L-phenylalanine, DL-phenylalanine, DL-tyrosine, sucrose and glyceric acid. Metabolic pathway analysis showed that the differential metabolites of tobacco leaves in flue-cured tobacco variety G80 at different maturation stages were significantly enriched in the phenylalanine metabolic pathway. Metabolites such as lignans, phenylpropanoic acid, flavonoids, and coumarins and their derivatives, which were accumulated in large quantities during the late stage of maturation, could improve the antioxidant capacity and aroma formation of the tobacco leaves.