The purification of recombinant modified human serum albumin-interferon α2b fusion protein(Ramsay-IFN-α2b) secretorily expressed by Pichia pastoris in high d. fermentation was studied. The recombinant Pichia pastoris strain PicZA/rmHSA-IFN-α2b/X33 was grown in 9-L fermentor and induced with methanol to express rmHSA-IFN-α2b. The supernatant was collected by centrifugation. RmHSA-IFN-α2b in the supernatant was purified by SP SepharoseTM FF, Blue SepharoseTM FF, and Q SepharoseTM FF in sequence. The existence of rmHSA-IFN-α2b in the processes was determined by SDS-PAGE. The purity of the final product was detected by HPLC and SDS-PAGE. The specific activity of rmHSA-IFN-α2b was determined by Cytopathic effect inhibition assay. The expression of rmHSA-IFN-α2b reached 0.421 g/L of supernatant. The rmHSA-IFN-α2b in the supernatant was purified in three steps. The first step was SP SepharoseTM FF, during which majority of yeast pigments and parts of other proteins were removed. The second step was Blue SepharoseTM FF, during which parts of yeast protein were removed. The third step was Q SepharoseTM FF, during which parts of yeast protein, solvent, and Endotoxin were removed. The purified rmHSA-IFN-α2b retaining its bioactivity was obtained. The final product exceeded 95% purity by HPLC and SDS-PAGE. The specific activity of rmHSA-IFN-α2b was 1.5*106 IU/mg. The total purification recovery rate was 25.3%. The purification route of rmHSA-IFN-α2b secretorily expressed by Pichia pastoris in high d. fermentation is successfully constructed. The purification process is simple, well reproducible, and suitable for large-scale production of rmHSA-IFN-α2b.