The invention discloses a natural biological fresh-keeping agent and a preparation method thereof, and relates to the technical field of fresh-keeping agents. The biological fresh-keeping agent is prepared from, by weight, 2-8 parts of Chinese bulbous onion, 4-10 parts of tea leaves, 5-9 parts of eutrema yunnanense, 3-7 parts of trichosanthes kirilowii maxim, 2-8 parts of rhizoma atractylodis macrocephalae, 4-10 parts of radix angelicae, 2-8 parts of gallnut, 0.5-3.5 parts of rosemary essential oil, 6-12 parts of wheat bran, 1-4 parts of glycerinum, 0.5-2.5 parts of tartaric acid, 10-20 partsof pullulan polysaccharide, 0.5-2.5 parts of Arabic gum, 0.2-2 parts of edible salt, 0.5-2.5 parts of calcium chloride, 0.5-3 parts of coconut oil glyceride and 60-100 parts of distilled water. Various components excellent in bacterium restraining and sterilizing effect are combined, antioxidants, film forming agents and the like are added for achieving a mutual synergistic effect, the effect of the fresh-keeping agent is improved, and the fresh-keeping time of the fresh-keeping agent for fruits and vegetables is prolonged. The fresh-keeping agent is prepared by adopting natural plant components according to a proportion and is free of toxicity and harm.