The invention discloses a serum-free medium for sheep ovarian cortical tissue in vitro culture, the culture medium includes basic culture medium and additives, the basic culture medium is a mixture of α-MEM and DMEM with a ratio of 6~10:1~4; the additives include the following components: 3~10 mg/mL bovine serum albumin, 3~10 μg/mL insulin, 1~5 mM glutamine, 0.10~0.50 mM sodium pyruvate, 2~8 ng/mL sodium selenite, 1~5 mM hypoxanthine, 10~200 μg/mL VC, 1~5 μg/mL transferrin, 50~200 IU/mL penicillin and 50~200 IU/mL streptomycin, the pH is 6.9~7.5. The invention solves the problem that the primordial follicles in ovarian cortical tissue cannot be cultured in vitro for a long time, which limits industrial development and teaching, and scientific research。
