A biocatalytic cascade reaction was designed for the stereoselective synthesis of optically pure 2-alkyl-1,3-diols employing two enzymes. The cascade process consists of two consecutive steps: a stereoselective diketone reduction and a hydroxy ketone reduction. Chiral diols were formed by the addition of ketoreductases in the same vessel, in high stereoselectivity and chemical yield, without the isolation of the intermediate β-hydroxy ketones.