Different sterilization doses of the electron beam (E-beam) will change the properties of biomaterials and affect their clinical application. Acellular porcine cornea (APC) is a promising corneal substitute to alleviate the shortage of corneal resources. The residual DNA was significantly reduced to 18.50 ± 3.19 ng/mg, and the clearance rate of α-Gal was close to 100% after the treatment with freezing–thawing combined enzyme, indicating that the decellularization was effective. The effects of different E-beam doses at 0, 2, 8, 15, and 25 kGy on the APC were studied. With the increase in irradiation dose, the transmittance, tensile strength, and swelling ratio of APC gradually decreased, but the resistance to enzymatic degradation was stronger than that of non-irradiated APC, especially at 8 kGy. The structure of APC was denser after irradiation, but the dose of 25 kGy could cause partial collagen fiber fracture and increase the pore size. The cell viability of the APC irradiated by 15 and 25 kGy were greater than 80%. After the implantation in rabbit corneas, there was no obvious neovascularization and inflammation, but the dose of 25 kGy had a more destructive effect on the chemical bonds of collagen, which made the APC easier to be degraded. The thickness of APC in the 25 kGy group was thinner than that in the 15 kGy group 1 year after surgery, and the epithelium grew more slowly, so the E-beam dose of 15 kGy might be more suitable for the sterilization of APC.