Abstract: An immortalized ?-1,3-galactosyltransferase gene knockout (GTKO) pig hepatocyte cell line, the preparation method and its application in preparing bioartificial liver and preparing medicine for treating liver failure. The immortalized GTKO pig hepatocyte cell line of this invention retains the main characteristics of the primary GTKO pig hepatocytes, including the function of urea synthesis and albumin synthesis. The cell line can be subjected to near-unlimited expansion culture in vitro, and can be used to study key molecular and drug intervention targets in xenograft rejection. When applied to bioartificial liver treatment, the immortalized GTKO pig hepatocytes can effectively solve the problem of xenogeneic hyperacute immune rejection, and reducing the use of immunosuppressive agents, prolonging the survival of transplant recipients and the time of normal liver function. Thus, the immortalized GTKO pig hepatocytes have important medical application prospects.