The invention relates to protein feed rich in small peptides and multi-enzyme strains and a preparation method thereof. The protein feed is prepared by fermenting soybean meal, cottonseed meal, rapeseed meal, bone meal, wheat bran, common salt, light calcium carbonate, water and strains in a fermentation bin. The preparation method of the protein feed comprises the following steps: mixing the soybean meal, the cottonseed meal, the rapeseed meal, the bone meal, the wheat bran, the common salt and the light calcium carbonate in percentage by weight; adding 20-30 percent of water; sending the mixture to an air steamer; adding high-pressure steam to the air steamer so as to increase the air pressure to 1-2 Mpa and the temperature to 140-200 DEG C; keeping the pressure for 4-6 minutes; momentarily releasing the mixture in the air steamer; sending the mixture to the fermentation bin; inoculating 3-5 percent of strains when the temperature of the mixture falls to 30-37 DEG C; fermenting for 72 hours at the temperature of 32-37 DEG C; discharging from the bin; and bagging to obtain a finished product. The product has strong disease-resistant function and low cost, increases the production of animal by-products by more than 20 percent, is nontoxic, harmless and pollution-free and can inhibit food-borne diseases represented in cancers and subhealth phenomena from spreading.