The invention discloses a preparation method of water-soluble active polysaccharide in boletus edulis, and an application thereof. The method comprises steps that: (1) boletus edulis is inoculated to a solid slant cultivation medium, such that slant seeds are obtained; (2) the cultivated slant is inoculated to a primary liquid seed cultivation medium, and is cultivated; (3) the cultivated primary liquid seeds are inoculated to secondary liquid seeds, and are cultivated; (4) the cultivated secondary liquid seeds are inoculated and fermented; (5) mycelium is separated from a fermentation broth, such that mycelium dry powder is obtained; (6) the mycelium dry powder is subject to ultrasonic lixiviation, such that liquid is concentrated; boletus edulis polysaccharide crude precipitate is collected; the boletus edulis polysaccharide crude precipitate is dissolved by using water; free protein is removed; the solution is concentrated, and is precipitated by using ethanol; the precipitate is dried, such that a protein-removed boletus edulis intracellular polysaccharide precipitate is obtained. The boletus edulis water-soluble active intracellular polysaccharide is applied in the preparations of medicines used for treating or preventing bacteria. The method provided by the invention is advantaged in simple method, convenient operation, low temperature, short time, and high efficiency. The prepared polysaccharide has good antibacterial property.