The invention discloses a preparation method of yeast beta-glucan. The preparation method comprises the following steps: adding 5-15 weight parts of brewer's yeast into 30-90 weight parts of enzyme liquor, adjusting the pH of the obtained solution to be 5-10, carrying out autolysis induction for 10-30 h under 45-65 DEG C water bath condition, and carrying out enzyme deactivation for 5-15 min under the 90-100 DEG C water bath condition, thus obtaining enzymatic hydrolysate; (2) centrifuging the enzymatic hydrolysate, and carrying out water washing for 2-4 times, thus obtaining sediment; (3) adding the sediment into 10-30 weight parts of acid liquor, carrying out acidolysis for 1-5 h under 65-125 DEG C, carrying out centrifugation, and carrying out water washing for 1-3 times, thus obtaining an acidolysis product; and (4) washing the acidolysis product by adopting alcohol for 1-3 times, carrying out filtering, and carrying out drying, thus obtaining the yeast beta-glucan. Compared with the prior art, the preparation method provided by the invention has the advantage that the product obtained by the preparation method provided by the invention is higher in purity; with the preparation method, the comprehensive utilization for the industrial by-product, namely, the waste beer yeast is realized, therefore, the utilization ratio and added value of the industrial by-product are increased, and thus the preparation method has great economic benefits and environmental protection significance.