The invention relates to gene engineering, and concretely discloses a CPP5-Cre fusion protein used for knocking out marker gene, and its application. The CPP5-Cre fusion protein comprises a short 5amino acid cell penetrating peptide CPP5 (KLPVM) and a site specificity recombinase Cre protein. F0 generation screening marker gene knocked-out transgenic pigs are obtained by carrying out screening marker gene knocked-out positive cloning obtained after the incubation of express-purified CPP5-Cre fusion protein and porcine fibroblast with the screening marker gene, and carrying out somatic cell cloning.