Porcine interferon-alpha (pIFN-alpha) could be used as the vaccine adjuvant to enhance the antiviral ability of porcine in swine industry. In here, a combinational strategy integrating codon optimization, multiple gene insertion, strong AOX1 promoter, and efficient secretion signal sequence was developed to obtain high-level secreted pIFN-alpha in Pichia pastoris GS115. The codon optimized pIFN-alpha shared 76% sequence identity with the original pIFN-alpha, which was inserted into the P. pastoris genome under AOX1(d1.2x201) Promoter and MF4I secretion sequence. Our results showed positive correlation between the mRNA and secreted protein levels with the copy numbers of genome-integrated pIFN-alpha gene in the recombinant P. pastoris strains. The recombinant opt-pIFN-alpha-6C strain bearing six copies of pIFN-alpha expression cassette produced the highest extracellular secretion of pIFN-alpha of 3.2 +/- 0.1 mg/mL in shake flask experiment, and 17.0 +/- 0.8 mg/mL in a 5 L high-cell-density cultivation after methanol induction of 84 h. The antiviral activity of secreted pIFN-alpha from the high-cell-density cultivation was determined to be approximately 2.8 +/- 0.9 x 10(9) IU/mL against the vesicular stomatitis virus (VSV) infected Madin-Darby bovine kidney (MDBK) cells. This strategy provided an efficient way to generate recombinant P. pastoris strains in a non-antibiotics-selection manner, which might also give general guidance for the heterologous expression of other proteins in P. pastoris.