The invention relates to a preparation method of recombinant horse interferon-alpha-1. According to the preparation method, a nucleotide sequence which is optimized by codon, and is used for coding horse interferon-alpha-1 is introduced into a yeast cell induced expression vector, and then is introduced into yeast cells for culturing and induced expression, and purification is carried out so as to obtain recombinant horse interferon-alpha-1, wherein preferably, the yeast cell induced expression vector is a secretory expression vector, after induced expression, a cell fermentation supernatant is obtained via culturing, and liquid phase column chromatography is adopted for purification of the cell fermentation supernatant. Design of the preparation method is skillful; recombinant horse interferon-alpha-1 with biological activity can be obtained via high expression at high purity, the recombination recombinant horse interferon-alpha-1, can be used to preventing houses from virus infection, and improving virus resistance of animals.