The invention discloses a beneficial microorganism antibiont-free feed and a preparation method of the beneficial microorganism antibiont-free feed. The preparation method comprises the following steps of: preparing a Lactobacillus acidophilus bacteria solution, a lactobacillus plantarum bacteria solution, a Saccharomyces cerevisiae bacteria solution and a Bacillus licheniformis bacteria solution respectively by carrying out secondary fermentation; then mixing the Bacillus licheniformis bacteria solution with cottonseed meal to carry out aerobic fermentation; mixing the fermented cottonseed meal, corn flour, beer slag and premixes uniformly, and adding a compound bacteria solution prepared from the Lactobacillus acidophilus bacteria solution, the lactobacillus plantarum bacteria solution and the Saccharomyces cerevisiae bacteria solution, mixing uniformly, carrying out constant temperature fermentation, thus obtaining the antibiont-free feed. The antibiont-free feed disclosed by the invention is not added with antibiotic medicines, is high in the content of the beneficial microorganism, has good attractant performance, palatability, and high nutrition digestion and absorption rate, increases the feed intake of animals, promotes the animal growth, enhances the level of animal health, and improves animal immune resistance to disease. The antibiont-free feed is applicable to breeding of antibiont-free livestock and poultry products, so that the breeding cost can be lowered, the economic efficiency is enhanced and the healthy development of aquaculture is promoted.