The invention relates to a preparation method of a bacteriocin biological fresh-keeping film, and is suitable for the field of fresh-keeping packaging of low-temperature foods. The preparation method comprises the following steps of using lactobacillus plantarum subsp.plantarum Zhang-LL (CGMCC No. 6936), activating, expanding and culturing, centrifuging to obtain a suspension, settling ammonium sulfate, dialyzing, and concentrating, so as to obtain a lactobacillus plantarum subsp.plantarum Zhang-LL solution; uniformly mixing the lactobacillus plantarum subsp.plantarum Zhang-LL solution and absolute ethyl alcohol, so as to obtain a coating solution; coating by a coating tester (ZY-TB), and naturally drying, so as to obtain the bacteriocin biological fresh-keeping film. The bacteriocin biological fresh-keeping film prepared by the preparation method has the advantages that the storage stability is good, and the bacteriostatic activity is at least maintained for six months or longer under the normal-temperature storage condition; the technology is simple, the cost is low, any toxic or side effect is avoided, the bacteria inhibiting effect is good, and the bacteriocin biological fresh-keeping film is suitable for industrial production.