Abstract: In order to explore the changes of sugar metabolism in different varieties of flue-cured tobacco, clarifying the molecular mechanism of the formation of tobacco leaf quality, the flue-cured tobacco varieties Qinyan 96, Yuyan 6 and K326 were taken as the sample materials to determine the changes of water-soluble sugar components (glucose, fructose, maltose, sucrose, reducing sugars, and total soluble sugars), key enzyme activities in sugar metabolic and gene expression changes at mature stage. The results showed that the trend of change in water soluble sugar content in the three varieties of flue-cured tobacco was basically the same. The contents of water soluble sugars (except sucrose) first increased and then decreased, reaching the peak when tobacco leaves matured. The sucrose content showed a bimodal fluctuation, and the sucrose content was the lowest when the tobacco was mature. The activity of SPS and AI enzymes contributed the most to the accumulation of water-soluble sugars in tobacco leaves from topping to proper maturity. NtINV played a major role in regulating sugar metabolism in tobacco leaves. When tobacco leaves were from ripe to overripe status, AI enzyme activity was mainly involved in tobacco sugar metabolism, while SS and SPS played an important role in sucrose accumulation. NtSS and NtSPS played a major role in regulating sugar metabolism. Under the same ecological environment and cultivation conditions, the content of metabolites in Yuyan 6 was enriched and had high water-soluble sugar content. The difference of SPS and AI enzyme activity may be an important reason for the different sugar components and content of tobacco leaves.