Abstract: To highly express secreted porcine interferon-beta (rPoIFNβ), the signal peptide was excised from the interferon-beta gene and the mature gene (mPoIFNβ) was cloned into the yeast-Escherichia shuttle vector pPIC 9K to construct secreting recombinant expressing plasmid of pPIC 9K-mPoIFNβ. The pPIC 9K-mPoIFNβ was linearized by SalⅠ and co-transformed with ssDNA into Pichia pastoris cells GS115 with LiCl. The GS115 was defective with histidine (His). The transformants were selected with MD culture plate and identified by PCR. And then, the multicopy recombinant Pichia pastoris strain was selected by G418 resistance. The selected strain could specifically secret 22kD and 26 kD mPoIFNβ proteins which was demonstrated by SDS-PAGE and Western blot. The mPoIFNβ proteins were about 29.4%–30.8% of total secreted proteins and the concentration was about 80 mg/L. The results of physicochemical property of the secreted protein indicated that the recombinant protein was insensitive to acid (pH2), partly sensitive to heat (56℃), and the antiviral activity could be neutralized by anti-PoIFNα serum not by anti-PoIFNγ serum. The antiviral activity of rPoIFNβ was tested by CPE50 method. The results showed that the rPoIFNβ was of high antiviral activity, which was 2.0×106U/mg against VSV in MDBK cells. The rPoIFNβ could suppress FMDV in IBRS-2 cells.