The invention relates to the technical field of blood culture bottle production, in particular to a reagent for blood culture bottle production and a preparation method thereof. The reagent comprisesbase solution, nutrient solution and fluorescent substance, wherein the base solution comprises the following raw materials in parts by weight: 800-1000 parts of silica gel RT601A, 50-150 parts of silica gel RT601B, and 2-4 parts of sodium hydroxide, the nutrient solution is composed of 13-17 parts of beef extract, 7-9 parts of agar, 8-12 parts of peptone, 3-7 parts of glucose, 3-7 parts of sodiumchloride, 0.5-1.5 parts of L-arginine, 2-4 parts of sodium benzoate, 1-2 parts of calcium lactate, 0.15-0.35 part of SPS, and 1.4-1.8 parts of Tris and 8-12 parts of fluorescent substances are included. The preparation method of the reagent comprises the following steps: S1, preparing solution: weighting raw materials according to the formula, sequentially adding the raw materials in to a crucible, heating to boiling, adding weighed agar, stirring, mixing, and continuously heating until the mixture is completely molten. According to the present invention, the preparation efficiency of the reagent is improved, and the diagnosis accuracy is significantly improved.