The invention discloses a method for preparing beta-carotene from Blakeslea trispora mycelium. The method comprises extraction and recrystallization process stages. A two-step recrystallization method is adopted in the recrystallization processing stage and comprises the steps that B1, total extracting liquid is concentrated till the concentration of beta-carotene is 5500-6500 mg/L, heat preservation and standing are carried out at the temperature of -5-10 DEG C, and then filtration is carried out; B2, the filtrate obtained in the step B1 is concentrated till the concentration of beta-carotene is 5500-6500 mg/L, heat preservation and standing are carried out at the temperature of 0-15 DEG C, and then filtration is carried out. Mycelium drying is not needed, wet mycelium is directly used for extraction, the process is simple, energy consumption is saved, only single organic solvent is used in the whole process, the production process is easy to control, the cost is low, the high purity and high yield of the product can be ensured at the same time, and the method is suitable for industrial production.