The invention relates to a preparation method of a microbial feed additive, which belongs to the technical field of feed additives. The preparation method comprises the following steps: inoculating aliquid culture medium with bacillus subtilis and lactobacillus, centrifugally concentrating to obtain thallus, preparing thallus sol by virtue of a sol-gel method, i.e. the microbial feed additive. The lactobacillus can improve the non-specific immunity of animals and can generate digestive enzymes such as amylase, lipase and protease and can generate bacteriocin to inhibit the growth of pathogenic bacteria, and the bacillus subtilis is high in resistance, resistant to high temperature, acid, alkali and high pressure, is good in tolerance to the particle feed processing, exists in a spore formin the storage process, does not consume the nutritional ingredients of the feed, can be rapidly revived in intestinal ducts (the revival rate is approximately 100 percent), and can generate variousamino acids and various digestive enzymes such as amylase, protease and lipase to consume oxygen in the intestinal ducts and to maintain the anaerobic environment, thereby inhibiting the growth of thepathogenic bacteria.