To explore the appropriate number of leaves for one-time harvesting of upper tobacco leaves in the tobacco-rice rotation region in southern Hunan, using flue-cured tobacco varieties Yunyan87 and Xiangyan7 as materials, a 3-level(5 leaves, 6 leaves, and 7 leaves) one-time harvesting experiment was designed. The SPAD values of leaves from different leaf positions were determined, and the economic traits and the quality of flue-cured tobacco were analyzed. The results showed that as the number of harvested upper tobacco leaves increased, the coefficient of variation of SPAD values and the maturity differences among leaves from different leaf positions increased. The proportions of positive group tobacco leaves, single leaf weight, leaf thickness, leaf mass per unit area, nicotine content, chlorogenic acid content, and smoking quality of the cured tobacco leaves decreased, while the proportion of variegated tobacco leaves and slightly green tobacco leaves, as well as the starch content and the ratio of reducing sugar to total sugar increased. Harvesting 5 tobacco leaves instead of 7 could increase the proportion of positive group tobacco leaves by 9.6% to 11.7%, increase the rate of high-grade tobacco by 9.1% to 21.2%, increase the appearance quality index by 10.1% to 10.6%, and increase the sensory quality index by 4.6%. The comprehensive effect indexes of Yunyan87 harvested with 6 leaves were 0.16% and 3.52% higher than that of Yunyan87 harvested with 5 leaves and 7 leaves, respectively. The comprehensive effect indexes of Xiangyan7 harvested with 6 leaves were 3.97% and 7.39% higher than that of Yunyan87 harvested with 5 leaves and 7 leaves, respectively. Based on the comprehensive quality and economic indicators of the cured tobacco leaves, the appropriate number of leaves for one-time harvesting of upper tobacco leaves in tobacco-rice rotation region of southern Hunan is 5 to 6 leaves for Yunyan87 and 6 leaves for Xiangyan7.