The invention discloses a method for preparing shiitake bioactive peptide. The method specifically comprises the following steps that (1) distilled water with the weight 6-12 times that of shiitake foot powder is added to the shiitake foot powder and the shiitake foot powder is dissolved; (2) ultrasonic disruption is conducted, 100 thousand to 200 thousand international units of cellulase are added to per kilogram of the shiitake foot powder, the pH value is adjusted to be 4.0 to 5.0, the temperature is adjusted to be 40 DEG C to 50 DEG C, and hydrolyzing lasts for 60 min to 120 min; (3) the shiitake foot powder solution is cooled to the indoor temperature, the pH value of the shiitake foot powder solution is adjusted to be 9.5 to 10.5 by sodium hydroxide, cold bath extraction is conducted for 20 min to 40 min at the temperature of 5 DEG C to 15 DEG C, supernate is extracted in a centrifugal mode, pressure reduction concentration is conducted, absolute ethyl alcohol is added until the concentration reaches 83% to 85%, standing is conducted for 60 min to 90 min at the temperature of 4 DEG C, centrifugation is conducted, precipitate is collected, and shiitake protein is obtained after drying; (4) the shiitake protein is dissolved in distilled water with the weight 40-60 times that of the shiitake protein, the pH value is adjusted to be 6.0 to 7.0, 0.2%-0.4% compound protease is added for hydrolysis, the temperature ranges from 45 DEG C to 55 DEG C, hydrolysis is conducted for 150 min to 200 min, enzyme deactivation is conducted, centrifugation is conducted, and filtrate is collected; (5) microfiltration is conducted through a filtering film of 0.22 micron, ultrafiltration is also conducted, peptide fragments with relative molecular mass ranging from 5 KD to 10 KD are collected, vacuum freeze drying is conducted, and therefore brown freeze-dried powder of the shiitake bioactive peptide is obtained. The prepared shiitake bioactive peptide has the good antioxidant activity in vitro and sobering activity.