The invention discloses a microbial fertilizer for improving activity of functional microbes and a preparation method of the microbial fertilizer, and belongs to the field of preparation methods of biological fertilizers. The method comprises the following steps: uniformly stacking organic waste, and performing ventilation fermentation to obtain a decomposed organic fertilizer; performing drying naturally, placing the dried material in a drying room, and performing sterilization at a high temperature; adding the functional microbes, a nutrient auxiliary agent and a conditioning agent, performing uniform adsorption, adjusting the humidity, and performing secondary fermentation; and performing granulation on the finished-product microbial fertilizer, performing air drying, and performing packaging. According to the method provided by the invention, after the sterilization operation is completed, the secondary fermentation is performed, inoculation is performed by using the functional microbes, so that a quantity of impurity microbes is reduced, and the dominant position of the functional microbes is improved; the nutrient auxiliary agent is added into the organic fertilizer, so thatthe survival activity of the microorganisms is improved, improvement of activity of the microorganisms is facilitated, respiratory metabolism to release heat of the microorganisms is promoted, and decomposition of the organic fertilizer is slowed down; and the method solves the problem that functional microbes of a current microbial fertilizer have a poorer survival effect and the microbial fertilizer has a shorter shelf life.