Nitrifier plays crucial role in improving soil ecological factors and regulates physiological function of crops. Evidences have suggested that biochar can be regarded as high quality energy to regulate the biochemical processes of crops. In this study, we investigated the effects of nitrifier cooperated with biochar on soil ecological factors and disease resistance of tobacco. A genetically engineered nitrifier delivered monoamine oxidase gene (MO) were constructed and developed to acquire the available nitrogen source in soil. The effects of biochar on activity of genetically engineered nitrifier delivered MO (gNitrifier-MO) were detected in soil. The physiological functions and disease resistance of tobacco were analyzed. Results showed that biochar enhanced the activity of gNitrifier-MO compared the soil treated by single gNitrifier-MO, resulting in the more production of nitrate nitrogen. We observed that gNitrifier-MO cooperated with biochar significantly increased the numbers of bacterium and nitrobacteria in the soil. Activity of gNitrifier-MO enhanced by biochar promoted nitrogen deposition or precipitation. In addition, gNitrifier-MO combined with biochar markedly improved the quality and the yield of tobacco. We also found that disease resistance of tobacco was improved through integrate microbial diversity and enrichment. Furthermore, gNitrifier-MO combined with biochar treatment increased expression levels of nonexpressor of PR and SR. NPIR-induced SIPK and WIPK expression and phosphorylation were up-regulated in tobacco plants infected with tobacco mosaic virus (TMV) cultured in gNitrifier-MO combined with biochar soil. SAR, PR-1α and POD in tobacco plants were increased in tobacco plants infected with tobacco mosaic virus (TMV) cultured in soil treated by gNitrifier-MO combined with biochar. In conclusion, these results indicate that gNitrifier-MO combined with biochar treatment improves disease resistance through regulation of NPIR-induced SIPK/WIPK pathway, suggesting gNitrifier-MO combined with biochar may be potential scheme for improvement of disease resistance of tobacco plant.