Abstract: The activity of nitrogen reductase and amylase and the accumulation of carbon and nitrogen compounds in the middle and upper leaves in the tobacco variety of 9601 during the mature stage were compared and analyzed with the chemical compositions and their coordination of flue-cured tobacco to study the effects of leaf population on the quality formation in flue-cured tobacco. The results showed that during the mature stage, the higher the leaf population, the faster the decreasing rate of NRA in middle-leaves and upper-leaves, and the lower the peak value of the activity of amylase; the higher the leaf population, the later the decreasing of NRA in upper-leaves and peak of starch accumulation, and the later changing to carbon metabolism mainly from nitrogen metabolism mainly; the tobacco leaves were closer to the high quality standards when the leaf population was 18-20.