This study aims to develop a cross-linked decellularized porcine corneal graft (cDPC) as a substitute for lamellar donor corneas and to evaluate the feasibility of using cDPC to treat fungal keratitis. The cDPC was prepared by decellularization, chemical crosslinking and γ-ray irradiation. Transparency, effectiveness of decellularization and biomechanical strength of cDPC were evaluated. The safety and efficacy of using cDPC to treat fungal keratitis were evaluated in the rabbit model. The transparency of cDPC was similar to that of a native porcine cornea (NPC), and no intact cells were observed in cDPC except for an insignificant amount of residual shrinking cellular nucleus. Compared to the NPC, the biomechanical strength of the cDPC was significantly increased. In the rabbit model of lamellar keratoplasty, the implanted cDPC reduced the incidence of corneal perforation, and also maintained transparency in majority. The results of this study suggest that the cDPC is capable of restoring the original transparency of cornea while effectively treating fungal keratitis. The cDPC is a highly promising ideal substitute for the donor human cornea.