Abstract: Flavonoid are important aroma precursors, which are controlled by many regulatory genes. Chalcone synthase gene (CHS), as one of the key gene of the flavonoid pathway, plays an important role in the synthesis of aroma precursors. The function missing or mutation of the CHS would affect the aroma formation. To explore the effects of CHS down regulatory expression and to provide technical information for breeding high aroma tobacco, we constructed the RNAi expression vectors by Gateway technology. Then the vectors were transformed into the ‘Dabaijin599’ by Agrobacterium-mediated method to form basis for further study in the aroma of CHS gene. Thirty of tobacco plants were obtained and 24 of them were identified as transgenic plants.