The invention relates to a preparation method of coix seed polysaccharide. The preparation method comprises the following steps of: crushing and screening coix seeds first, and carrying out degreasing, water bath extraction, centrifugation, enzymolysis, concentration, alcohol precipitation and vacuum freeze drying and precipitation to obtain crude coix seed polysaccharide; and dissolving the prepared crude coix seed polysaccharide to obtain a crude coix seed polysaccharide solution, carrying out deproteinization, decoloration, dialysis and ion exchange chromatography on the crude coix seed polysaccharide solution to obtain purified coix seed polysaccharide, and then carrying out vacuum freeze drying to obtain white powder, namely the coix seed polysaccharide. According to the preparation method disclosed by the invention, the crude coix seed polysaccharide is extracted by using a water extraction and alcohol precipitation method, the crude coix seed polysaccharide is subjected to deproteinization, decoloration, dialysis and ion exchange chromatography to obtain the purified coix seed polysaccharide, and then, the purified coix seed polysaccharide is subjected to vacuum freeze drying to obtain the white powder, namely the coix seed polysaccharide; and therefore, the preparation method is simple and the coix seed polysaccharide is high in yield.