The present invention discloses a preparation method for prion protein gene knockout domestic animals. The process is characterized by: knocking off the prion protein gene of domestic animals on a cellular level; and preparing prion protein gene knockout domestic animals through somatic cell cloning method. The function of the prion protein of domestic animals is inactivated. The functional prion protein is not expressed, thus the domestic animals can resist to relative disease of prion protein, such as scrapie, bovine spongiform encephalopathy (mad cow disease) and the like.