The invention relates to a grape biological fresh-keeping agent which comprises 1-8 g of sodium chloride, 1-6 g of calcium chloride, 3-9 g of chitosan, 0.1-0.5 g of nisin, 1-10 g of beta-1,3-glucanase, 15-40 g of B-CHD bacillus fermentation liquor, and 1 L of normal temperature sterile water; the preparation method comprises the following steps: adding 1-8 g of sodium chloride, 1-6 g of calcium chloride, 3-9 g of chitosan, 1-10 g of beta-1,3-glucanase, 0.1-0.5 g of nisin with a concentration unit of 75 units/150 ul into 1 L of normal temperature sterile water, stirring to completely dissolve; then adding 15-40 g of B-CHD bacillus fermentation liquor. The advantages of the invention are that the product is free of pollution, low in cost, safe, and nutritious, has multifunctional characteristics of obviously reducing grape fruit decay and threshing, preventing oxidation and coloration, and preventing fiber decomposition and softening, is suitable for storage and fresh keeping after grape picking, and is an economical and ideal biological fresh-keeping agent.