Abstract: To explore the effects of maturity, variety and their interaction on polyphenols in flue-cured tobacco, the experiment was designed using three different flue-cured tobacco varieties (K326, Yuyan 11, Yunyan 87) and three different harvesting methods (unripe, ripe and mellow) in a three-level random block design of two-factors. The contents of total phenol, chlorogenic acid, rutin and scopoletin in tobacco leaves were determined. The results showed that there were significant differences in polyphenol contents among different maturities, varieties and combinations. Polyphenol content was mainly affected by maturity, followed by variety. The contribution rates of maturity, variety and their interaction to total phenol, chlorogenic acid, rutin and scopoletin contents were different. The contribution rates of maturity to total phenol, chlorogenic acid, rutin and scopoletin contents were 52.93%, 55.83%, 48.38%, 73.58% respectively, and the content of each polyphenol in ripe harvesting tobacco leaves was the highest. The contribution rates of varieties were 32.31%, 31.57%, 32.97%, 16.04%, and the content of each polyphenol in K326 was the highest. The contribution rates of maturity and variety interaction were 14.64%, 12.32%, 18.29%, 9.43%. K326 and Yuyan 11 have higher contents of total phenol, chlorogenic acid, rutin and scopoletin in ripe harvests. Yunyan 87 has higher contents of total phenol, chlorogenic acid and rutin in mellow harvests and has the highest content of scopoletin in ripe harvests. Harvesting at appropriate maturity for different flue cured tobacco varieties is an important and controllable approach to improve content of polyphenols in tobacco leaves.