The invention belongs to the technical field of mushroom deep processing, and discloses a biological enzymolysis mushroom extract and a preparation method thereof. The preparation method comprises thefollowing steps that raw materials are pretreated, enzyme deactivation is carried out, and after enzymolysis is carried out, cooling is carried out to reach the normal temperature so as to obtain hydrolysate; secondary enzymolysis is carried out, specifically, 5'-phosphodiesterase is added, the pH value is adjusted, heating is carried out to reach 50-60 DEG C, reaction is carried out for 1.5-2.5hours, heating is carried out to reach 95-100 DEG C, the temperature is kept for 10-20 minutes, enzyme deactivation is carried out, after enzymolysis is carried out, cooling is carried out to reach the normal temperature, filtering is carried out through a 60 to 80-mesh screen or centrifugal separation is carried out so as to obtain enzymatic hydrolysate; blending is carried out, specifically, a multi-enzyme enzymolysis mushroom extracting solution, beta-cyclodextrine and edible salt are uniformly mixed; and drying is carried out, specifically, spray drying is carried out on the mixed materials so as to obtain the mushroom extract. According to the mushroom extract and the preparation method thereof, a multi-enzyme enzymolysis technology is adopted to optimize extraction of mushroom flavouring materials, through multi-enzyme enzymolysis, the time can be saved, the energy consumption can be reduced, and the production efficiency can be improved; and the staged enzymolysis conditions aremild and are easy to control, and the obtained enzymatic hydrolysate contains rich amino acid, nucleotide, lentinan and other active ingredients.