The invention discloses a preparing method of lactobacillus acidophilus, which comprises the following steps: adopting liquid deep layer ferment technique to do two-step ferment culture manufacturingthrough inoculating torularhodin and lactobacillus acidophilus; inoculating torularhodin at 28-33 deg. c for 4-30h; heating to 40-60 deg. c to insulate 1-18h; making technique autolyze to form basic nourishing condition of lactobacillus acidophilus; cooling to 34-39 deg. c; inoculating lactobacillus acidophilus; cooling to 34-39 deg. c; inoculating lactobacillus acidophilus; breeding to culture for 15-100h; canning; modulating to obtain the product.