Abstract: The objectives of this study were to investigate the effects of the number of unusable fresh leaves removed on neutral aroma composition of flue-cured tobacco. A field experiment with four treatment level (0, 2, 3, and 4 leaves removed) was carried out by using Yunyan 97. The results indicated that the content of carotenoid and neophytadiene in upper leaves significantly increased when two leaves removed, but the content of phenylalanine, vembratriendiol, varotenoid in middle leaves significantly decreased. The content of phenylalanine, carotenoid, neophytadiene in middle leaves, and the content of phenylalanine, maillard reaction products, cembratriendiol in upper leaves significantly increased when 3 leaves removed, but the content of maillard reaction products in middle leaves decreased significantly. The content of maillard reaction products and cembratriendiol in middle leaves significantly increased when 4 leaves removed, but the phenylalanine and carotenoid in upper leaves decreased. The total content of neutral aroma component in flue-cured tobacco was significantly improved due to removal of the fresh leaves. The content in middle leaves from high to low was: 3 leaf removal > 3 leaf removal > 2 leaf removal > no removal. For upper leaves, the order was 2 leaf removal > 3 leaf removal > 0 removal > 4 removal. Therefore, we suggest the 3 leaf removal is the best option.