Abstract: In order to explore the effects of nitrogen supply levels on the activities and gene expression of key enzymes of sugar metabolism, and the content of carbohydrate, and the relationship among these three elements in flue cured tobacco, a two factor experiment with nitrogen levels and varieties was carried out. The results showed that the activity of sucrose synthase increased with the increase of nitrogen application, and the difference was significant among treatments. The activities of sucrose phosphate synthase, invertase and amylase increased with the increase of nitrogen application on the 45th and 60th day after planting. In contrast, on the 80th day after planting, the activities of invertase and amylase were higher in low amount of nitrogen supply treatment than those of higher amount of nitrogen supply treatment, while there were no significant difference in enzyme activities among the variety treatments. The amount of nitrogen supply also had significant effects on the expression levels of sucrose synthase, sucrose phosphate synthase and invertase genes. There were some difference in the gene expression levels between varieties under the same nitrogen supply treatment. The contents of total sugar and reducing sugar in tobacco leaves were less affected by the amount of nitrogen applied. However, the contents of starch increased with the increase of nitrogen application. There was no obvious difference in the contents of carbohydrate of varieties tested under the same amount of nitrogen treatment. The results of correlation analysis showed that the activities of these enzymes were extremely significantly or significantly correlated with the starch content in tobacco leaves at each growth stage. The expression of sucrose phosphate synthase gene was negatively correlated with the starch contents of tobacco leaves on the 45th day after planting, while the expression of sucrose synthase gene was significantly positively correlated with reducing sugar and total sugar on the 60th and 80th day after planting respectively. Our results suggest that the amount of N application has a significant effect on activity and gene expression of key enzymes in tobacco sugar metabolism. Sugar content was determined by the activity and gene expression of sucrose synthase and sucrose phosphate synthase.