The invention discloses a method for preparing biotin-intermediate lactone by enzyme chiral oxidation. In the existing method for asymmetric hydrolysis synthesis of the lactone by utilizing a pig liver esterified enzyme, the e.e. value of the obtained lactone is low and the lactone can not meet the needs on the biotin optical activity. The method adopts horse liver tissues as raw materials to prepare dry acetone powder of horse liver alcohol dehydrogenase; 1,3-dibenzyl-4,5-cis-bis(hydroxymethyl)-2-imidazolinone is taken as a biological transformation substrate, the dry acetone powder of the horse liver alcohol dehydrogenase is a chiral oxidase, and the biotin-intermediate lactone is synthesized through the enzyme catalytic oxidation reaction. The horse liver alcohol dehydrogenase has the advantages of specificity, chiral catalytic selective oxidation, mild reaction conditions, and the like, the e.e. value of the obtained lactone is not less than 95 percent, and the lactone can meet the needs on the biotin optical activity.