The invention relates to the technical field of medical biomaterials, in particular to a culture medium, a corneal stromal slice prepared by the culture medium and a preparation method. The medium provided by the invention is a Dulbecco's modified eagle medium/F12 (DMEM/F12) medium containing a porcine corneal extract, Y-27632, insulin-transferrin-selenium (ITS), fibroblast growth factors (FGF), ascorbic acid and fetal bovine serum. According to the medium, by selecting suitable active components and proportion, a corneal stromal cell and corneal stromal slice prepared by employing the mediumhave the functions of maintaining the corneal stromal cell phenotype, promoting proliferation, improving vitality, resisting fibrosis and resisting apoptosis, and the like in comparison with a cornealstromal cell and corneal stromal slice prepared by a traditional medium. The corneal stromal slice disclosed by the invention is prepared by taking a corneal lens as a raw material and culturing thecorneal lens by employing the medium; and compared with animal-derived corneal repair material, the corneal lens has good biocompatibility and no antigenicity, can be used as substitutes of various donor materials for corneal transplantation, and can be accepted by the majority of patients and is clinically applied for long.