The invention belongs to the technical field of biological fertilizer preparation, and in particular relates to a preparation method of a bio-fertilizer for scutellaria baicalensis planting. The preparation method comprises the following steps: S1, raw material preparation: sequentially weighing the following raw materials in parts by weight; 6-10 parts of straw, 12-16 parts of soybean meal, 8-12parts of cottonseed meal, 4-8 parts of dry chicken manure, 6-10 parts of cassava residues, 6-10 parts of sugar residues, 5-9 parts of plant ash, 3-7 parts of peanut shells, 2-6 parts of sawdust, 42-46parts of rice husk powder, 2-6 parts of bacillus subtilis, 42-46 parts of bacillus mucilaginosus and 22-26 parts of peanut rhizobium. S2, crushing: adding straw, soybean meal, cottonseed meal, dry chicken manure, cassava residues, sugar residues, plant ash, peanut shells, sawdust and rice husk powder in the step S1 into a crusher for crushing treatment. The process for preparing the bio-fertilizer is simple, the fertilizer efficiency of the bio-fertilizer is good, and the requirement for scutellaria baicalensis planting is met.