Abstract: In order to evaluate the spatiotemporal dynamics of soil microbes and enzyme activities and tobacco output in tobacco-planting upland under no-tillage system, a field experiment with 3 treatments (no-tillage, tillage with no-hilling, and conventional tillage) was carried out. The results showed that (1) Among the soil macrobiotics, bacteria was the most, then was actinomycetes, and fungus was the least. The quantity of bacteria and microorganism activity increased firstly, then decreased during the tobacco growth period, which had maximum values at tobacco maturity. The quantity of soil microorganism decreased with soil depths, and the influence of no-tillage on the quantity of soil microorganism occurred mainly in 0 -5 cm surface soil. (2) Invertase activity increased during the later period of tobacco growth, the activity of urease and phoshatase were high at resettling stage, then decreased, and catalase activity increased firstly, then decreased. The effect of no-tillage on soil enzyme activity occurred mainly in 0-5 cm surface soil. This influence decreased with soil depths. (3) The no-tillage planting had not significant influences on yield, output value, average price of tobacco leaves and the ratio of superior tobacco leaves. Therefore, no-tillage system is worthy to be popularized in upland in western Hunan.