The invention discloses a preparation method and application of vinasse alcohol-soluble peptide, and belongs to the technical field of white spirit production. The method comprises the following steps: crushing vinasse, performing pretreatment by combining cellulose and amylase, performing hydrolysis by combining papain with polypeptide hydrolase or neutral protease, and after two-step hydrolysis, centrifuging and concentrating vinasse enzymatic hydrolysate to obtain vinasse crude peptide; and carrying out precipitation extraction, centrifugation, concentration, resin purification and nanofiltration interception grading on the vinasse crude peptide by using alcohol with a specific concentration to obtain a vinasse target alcohol-soluble polypeptide solution, concentrating and recovering ethanol, and drying to obtain the target product. The purity of the alcohol-soluble polypeptide prepared by the method is higher than 80%, the alcohol-soluble polypeptide is good in solubility, stable and free of precipitation after being added into a white spirit system, the molecular weight is smaller than 3000Da, and the alcohol-soluble polypeptide has potential biological activity and endows the product with dual characteristics of flavor and function.