The present invention provides a method for preparing yeast beta-D-glucan using a solubilization technology based on molecular assembly, comprising the following steps: (1) micro-fluidizing an enzymatic hydrolysate of yeast cell walls at 70 to 200 MPa, and then centrifuging to obtain a precipitate; (2) resuspending the precipitate obtained in step (1) with a ionic liquid, then dispersing to obtain a solution; wherein the ionic liquid is 1-ethyl-3-methylimidazolium acetate or 1-allyl-3-methylimidazolium chloride; (3) centrifuging the solution obtained in step (2), and then adding ethanol, centrifuging and collecting a precipitate; (4) resuspending the precipitate obtained in step (3) with water, then centrifuging and collecting a supernatant. Preferably, the method further comprises (5): spray drying the supernatant obtained in step (4) to obtain a yeast beta-D-glucan powder. The yeast beta-D-glucan obtained in the present invention has high purity and good solubility, which advantageously enlarges its application range.