The invention discloses a serum-free culture solution for in-vitro culture of sheep ovarian cortex tissue. The culture solution comprises a basic culture solution and an additive, wherein the basic culture medium is a mixed solution of alpha-MEM and DMEM; the additive comprises the following components: 3-10 micrograms / mL bovine serum albumin, 0.10-0.50mM sodium pyruvate, 1-5mM glutamine, 1-5mM hypoxanthine, 3-10 micrograms / mL insulin, 1-5 micrograms / mL transferrin, 2-8 nanograms / mL sodium selenite, 10-200 micrograms / mL VC, 50-200IU / mL penicillin and 50-200IU / mL streptomycin, and the pH valueis 6.9-7.5. Through the synergistic effect of the components, the proliferation speed of interstitial cells is inhibited, the proliferation of granular cells is promoted, the defect that the three-dimensional environment of original follicle development cannot be maintained due to tissue collapse caused by excessive proliferation of the interstitial cells in the culture process of the ovarian cortex tissue is overcome, the growth and development condition and survival time of original follicles in the ovarian cortex tissue in vitro are effectively improved, meanwhile, the influence of unknowncomponents in serum on research is avoided, and the serum-free culture solution has a good application prospect.