The invention discloses a preparation method of a microorganism active tobacco seedling growing base material. The preparation method comprises the following steps: activating EM bacteria; preparing a tobacco seedling growing base material activating agent; and fermenting and preparing the microorganism active tobacco seedling growing base material which is directly used for floating type seedling growing. By sufficiently expressing material properties of a microorganism bacterial agent (EM bacteria) (capable of optimizing base material properties, promoting rooting and reducing diseases and insect pests), organic materials (capable of improving nutritional requirements of tobacco seedlings and promoting expanding propagation of microorganisms) and biomass carbon (with abundant micro-pore structures, large specific surface area, high pH and strong adsorption capability and anti-decomposition capability); the three materials above are organically fused with peat, vermiculite, perlite and the like by utilizing a fermentation engineering technology to prepare the seedling growing base material which has the characteristics of high microbiological activity, capability of continuously and effectively supplying fertility, and good physicochemical environment for development of tobacco seedlings; the seedling growing base material is successfully used for the floating type seedling growing of tobaccos.