The invention discloses a preparation method of a chiral separation membrane. The preparation method of the chiral separation membrane comprises the following steps: soaking a polymer membrane in an aqueous solution containing metal ions; taking out the polymer membrane a period of time later; transferring the polymer membrane into the aqueous solution containing a reducing agent; vibrating to enable the metal ions which are adsorbed to the surface of the membrane to be converted into metal elements; soaking the membrane loaded with metal in a chiral molecular solution which contains thiol anddiphenol structures; taking out the membrane a period of time later; and washing chiral molecules which are not adsorbed with deionized water to form the chiral separation membrane. The preparation method of the chiral separation membrane is simple and efficient, conditions are gentle, and large-scale application can be realized.