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Introduction: The aim of this study was to develop a novel decellularization method in order to obtain an ideal scaffold with good biocompatibility. Methods: The porcine corneas were treated with human serum for 5 days or serum-electrophoresis respectively. The electrophoresis (100 V/cm) was performed in sterilized buffer containing 40-mM tris base, 18-mM glacial acetic acid, and antibiotics for 1 h at 4°C. The properties of artificial corneal scaffolds were characterized by morphological and histological examinations. The biocompatibility and biological safety were examined by subcutaneous implant test and lamellar keratoplasty. Results and conclusions: The transparency and appearance of serum-electrophoresis acellular porcine corneal matrix were better than serum acellular porcine corneal matrix. DNA and α-gal in serum-electrophoresis acellular porcine corneal matrix were more efficiently removed than those in serum acellular porcine corneal matrix (p < 0.05). The subcutaneous and corneal implantation experiments showed serum-electrophoresis acellular porcine corneal matrix had better biocompatibility compared to serum acellular porcine corneal matrix (p < 0.01). This novel serum-electrophoresis decellularization method may be valuable for preparation of xenogenic corneal tissue for clinical application.