The invention relates to a preparation method of a fertilizer containing microorganisms. The preparation method comprises: adding dried plant residues, an acid-alkali adjusting agent, distilled water, a culture medium and microorganisms into a fermentation tank, fermenting at a certain temperature, carrying out pH value adjustment and cooling on the product after fermenting for a certain time, storing for 3-5 days in the fermentation tank, adding slaked lime, adjusting the pH value, continuously adding the culture medium and the beneficial microorganism to the fermentation tank, fermenting at a certain temperature, carrying out pH value adjustment and cooling on the product after the fermenting, storing for 3-5 days in the fermentation tank, repeating the steps one time, carrying out air drying on the product, and adding to a fertilizer to obtain the fertilizer containing the microorganisms. According to the present invention, the natural ecological environment of the soil is simulated, and the three fermentations are performed, such that the fermented microbial agent can quickly adapt to the environment after being put into the soil so as to ensure the survival rate.