Type III interferons (IFN–λs) exhibit potent antiviral activity and immunomodulatory effects in specific cells. Nucleotide fragments of the bovineifn–λ(boifn–λ) gene were synthetized after codon optimization. Theboifn–λgene was then amplified by splicing using overlap extension PCR (SOE PCR), resulting in the serendipitous acquisition of the mutated boIFN–λ3V18M. The recombinant plasmid pPICZαA–boIFN–λ3/λ3V18Mwas constructed, and the corresponding proteins were expressed inPichia pastoriswith a high–level extracellular soluble form. Dominant expression strains of boIFN–λ3/λ3V18Mwere selected by Western blot and ELISA and cultured on a large scale, and the recombinant proteins purified by ammonium sulfate precipitation and ion exchange chromatography yielded 1.5g/L and 0.3 g/L, with 85% and 92% purity, respectively. The antiviral activity of boIFN–λ3/λ3V18Mexceeded 106U/mg, and they were neutralized with IFN–λ3 polyclonal antibodies, were susceptible to trypsin, and retained stability within defined pH and temperature ranges. Furthermore, boIFN–λ3/λ3V18Mexerted antiproliferative effects on MDBK cells without cytotoxicity at 104U/mL. Overall, boIFN–λ3 and boIFN–λ3V18Mdid not differ substantially in biological activity, except for reduced glycosylation of the latter. The development of boIFN–λ3 and comparative evaluation with the mutant provide theoretical insights into the antiviral mechanisms of boIFN–λs and provide material for therapeutic development.