Abstract: It has been proved that rational application of sesame cake fertilizer collaborated with chemical fertilizer could increase the output and quality of tobacco leaves. However, the responses of soil biochemistry properties on the application of sesame cake fertilizer were still not clear. A potted plant experiment was carried out on a loam soil cultivated with tobacco, in order to investigate the effects of sesame cake fertilizer on soil enzyme activities and microbial C and N. Four treatments were applied: no fertilizer (CK), single application of chemical fertilizer(F), single application of sesame cake fertilizer(C) and combined application of sesame cake fertilizer with chemical fertilizer treatment (CF, organic N and mineral N respectively accounted for a half of total N). The results indicated that the sesame cake fertilizer could promote the enzyme activities and increase microbial C and N contents in soil. Comparing with single application of chemical fertilizer, combined application of sesame cake fertilizer with chemical fertilizer could promote lipase, urease and invertase activities by 24.85%-60.00%, 23.08%-39.46% and 4.17%-56.21%, and increase soil microbial C and N contents by 79.88%-97.14% and 29.73%-74.96% respectively. The promotion of enzyme activity was of great advantage to the turnover of organic matter and provision of nutrition to meet the needs of tobacco's regular growth. The dynamic changes of microbial C and N content were different at different growth duration. Microbial C content reached the peak at the budding stage of flue-cured tobacco; however, the peak of microbial N content appeared at the rosette stage. This showed that inorganic N content was higher at the incipient stage of plant growth which resulted from the fixation of soil inorganic N by microorganism. Microorganisms may release N to meet the need of flue-cured tobacco at the late stage and microbial N was reduced markedly at mature stage. Therefore, Microbial N played an important role in the synchronization of soil N provision with the growth of flue-cured tobacco. We could conclude that the rational application of sesame cake fertilizer mixed with chemical fertilizer could balance nitrogen alimentation, which gave rise to the improvement of the quality of flue-cured tobacco leaves.