Abstract: Biochar is one of the research hotspots in the field of the agroforestry waste utilization. A field experiment was carried out to investigate the effects of different amounts of tobacco stem biochar (0, 1, 10, 50 t·hm^-2) on soil micro-ecology and physiological properties of flue-cured tobacco. The results showed that soil water content (SWC) increased at all tobacco growth stages as the amounts of biochar applications increased. There were significant differences of SWC between the treatment of 50 t·hm^-2 and other treatments at the period of tobacco vigorous growth. As the application of biochar increased, the total soil porosity and capillary porosity increased, while soil bacteria, actinomyces, fungi amount increased firstly and then decreased. The amount of soil bacteria, actinomyces, fungi reached the maximum at the treatment of 10 t·hm^-2. Soil respiration rate (SRR) at earlier stage increased with the increase of biochar application. Compared with the control, SSR under biochar treatments increased by 7.9%-36.9%, and there were significant differences of SRR between high biochar application treatments (50 t·hm^-2 and 10 t·hm^-2) and the control.  Biochar improved leaf water potential, carotenoid and chlorophyll contents. Meanwhile, the dry mass of root, shoot and total dry mass under biochar application were higher than that of the control. These results indicated that the biochar played active roles in improving tobacco-planting soil micro-ecology and regulating physiological properties of flue-cured tobacco.