The invention provides a fertilizer for cultivation of tobacco. The fertilizer consists of the components by weight as follows: 0.5-1.5 parts of a composite microbial agent, 3-7 parts of an activating agent, 3-7 parts of binder, and 86-92 parts of nitrogen, phosphorus and potassium fertilizer. The effective components of the composite microbial agent are Bacillus licheniformis, Bacillus pumilus, Brevibacillus laterosporus, Bacillus megatherium and Silicate bacteria. The invention further provides a preparation method and an application of the fertilizer. The product provided by the invention can remarkably improve crumb-structure of soil for cultivation of tobacco, and microflora distribution and micro-environment of the soil. A plenty of derivatives are generated, thereby chelating or blocking damage of heavy metals, and absorption and assimilation of tobacco to nutrient elements are improved, the drought resisting and disease resisting abilities of tobacco are enhanced, damage of diseases and insects caused by continuous cropping of tobacco in successive years are effectively prevented, and in particular, tobacco mosaic virus and brown spot disease are specially prevented and treated. According to the invention, the raw materials are low in cost and easy to obtain and the preparation and using methods are simple and easy to operate.