Abstract: The present disclosure provides a yeast mannan-oligosaccharide, a preparation method and a use thereof. The preparation method comprises the following steps: step S1, mixing the yeast cell wall with water to obtain a mixed solution; step S2, adjusting the pH of the mixed solution to 4-9, and carrying out a first enzymolysis with a protease to form a first enzymatic hydrolysate; secondly, subjecting the first enzymatic hydrolysate to a second enzymolysis with a polysaccharidase to obtain a second enzymatic hydrolysate; and step S3, subjecting the second enzymatic hydrolysate to centrifugal separation so as to obtain a supernatant and a centrifugal heavy phase, filtering the supernatant with an ultrafiltration membrane, the retentate being the yeast mannan-oligosaccharide, the permeate being the yeast extract, and the centrifugal heavy phase being the yeast glucan.