Abstract: A field experiment was conducted to study the effect of biochar on carbon (C) and nitrogen (N) conversion under different nitrogen application rates in tobacco-planting soil, and select the optimum amount of N application. Five treatments were set as: no N and no biochar (CK), and 1600 kg/hm2 biochar with four nitrogen levels: 0 kg/hm2(N0), 37.5 N kg/hm2 (N1), 52.5 N kg/hm2 (N2) and 67.5 N kg/hm2 (N3). The effect of biochar and nitrogen fertilizer on key enzyme activities of C and N cycle and the available nutrients was analyzed. The results showed that the activities of soil urease and sucrose were improved significantly under the application of biochar and nitrogen fertilizer, and the content of soil available nitrogen was significantly increased, the highest content of soil available nitrogen was 261.86 mg/kg under N3 treatment, while, there was little effect on soil available phosphorus content. The nitrogen application could increase the content of soil available potassium 60 days after tobacco transplanting and the content of soil available potassium increased first and then decreased with the increase of N application. What’s more, biochar and nitrogen fertilizer application improved microbial biomass carbon and microbial entropy. The highest content of microbial biomass carbon and microbial entropy were 355.00 mg/kg and 3.01% under N3 and N2 treatment, respectively. Generally, the combination application of biochar and 67.5 kg/hm2 N is the optimum practice in central Henan Province.