The invention discloses a method for preparing high-anti-fatigue-action soybean peptide determined by a microcalorimetric method and an anti-fatigue action evaluation experiment, and belongs to the technical field of soybean deep processing. The optimal protease for preparing the high-anti-fatigue-action soybean peptide is alkaline protease by adopting the microcalorimetric method according to the Michaelis constant comparison of an enzymatic hydrolysis reaction, and a product anti-fatigue-action evaluation experiment is combined to obtain the method for preparing the high-anti-fatigue-action soybean peptide. The method can be used for effectively improving the anti-fatigue function of a product. Compared with an anti-fatigue soybean peptide produced by a conventional process, high-anti-fatigue-action soybean peptide has the advantages of remarkably improving the exercise tolerance of a body, remarkably reducing accumulation of blood lactic acid after body exercise, remarkably increasing the reserve level of hepatic glycogen and remarkably improving the anti-fatigue capability of a body. The high-anti-fatigue-action soybean peptide has the advantages of no pollution, low cost, simple process, remarkable anti-fatigue function, good water solubility, safety, no toxicity, no side effect and the like, has a relatively high popularization value, can be used for generating relatively high economic benefit and social benefit, and has a broad prospect.