The application of sesame cake fertilizer can improve significantly the yield and quality of tobacco in Henan, but its wide use in tobacco planting is hindered by the high cost, however rapeseed cake fertilizer is more abundant and cheaper, and is possibly used to partly replaced sesame cake fertilizer for tobacco planting. In this study, the different ratios of rapeseed and sesame cake fertilizers were designed and their effects on microbial flora and biomass, enzyme activities and nitrogen conversion were studied in order to determine the reasonable ratio of the two cake fertilizers for fertilizing soil and improving tobacco quality. The results showed that compared with other treatments, the treatments of T5 (inorganic fertilizer + 30% rapeseed cake fertilizer + 70% sesame cake fertilizer) and T6 (inorganic fertilizer + 100% sesame cake fertilizer) had better microbial proliferation in the resettling, vigorous growing and budding stages, but the treatments of T2 (chemical fertilizer + 100% rapeseed cake fertilizer) and T3 (chemical fertilizer + 70% rapeseed cake fertilizer + 30% sesame cake fertilizer) had more microorganisms than those other treatments in the mature stage. The microbial biomass carbon reached the peak in the budding stage, but the microbial biomass nitrogen and phosphorus reached the peaks in the resettling stage. The activities of urease, protease, phosphatase and invertase were all significantly increased in the vigorous growing stage except for catalase, and the treatments of T5 and T6 had higher enzyme activities than other treatments. The treatments of T5 and T6 were more consistent with nitrogen nutrition characteristics in high-quality tobacco production and no significant difference between the two treatments. The treatments of T2 and T3 had slow nitrogen mineralization which was not satisfied with the demand for nitrogen absorption by tobacco. Therefore, the ratio of 30% rapeseed cake fertilizer + 70% sesame cake fertilizer is recommended to replace 100% sesame cake fertilizer in tobacco planting because it can save the cost, effectively improve the soil biological characteristics, balance the nitrogen nutrition and improve the quality of tobacco.