The invention relates to the field of probiotics, in particular to a preparation method of probiotic granules being resistance to acid and bile salt. The preparation method comprises the following steps of S1, uniformly mixing probiotic bacterial sludge with a protective agent, and performing slow stirring to obtain an emulsion; S2, adding prepared colloid liquid to the obtained emulsion, uniformly mixing and stirring the obtained emulsion with the colloid liquid, and performing slow stirring to obtain mixing liquid; S3, slowly injecting the obtained mixing liquid into a calcium chloride solution, performing a crosslinking reaction, after the mixing liquid is completely injected, performing high-speed shearing and stirring, and then performing standing to obtain mixed bacteria starch; andS4, performing freeze drying on the obtained mixed bacteria starch to obtain solids of which the water content is lower than 5%, and grinding and granulating the obtained solids by a grinding and granulating machine to obtain 20-50-mesh probiotic granules, wherein the protective agent comprises trehalose, glycerine and modified starch, and the colloid liquid comprises sodium alginate. The probiotic granules obtained through the preparation method disclosed by the invention have favorable acid resistance and bile salt resistance, and when the probiotic granules achieve intestinal tracts throughstomachs, the activity can be maintained to a great extent.