AIM: Interferon α2b (IFNα2b) and thymosin α1 (Tα1) exhibit synergic effects in the treatment of hepatitis B and hepatitis C when used together. For developing a fusion protein drug, fusion proteins of IFNα2b and Tα1 linked by different lengths of (G4S)n (n=1-3) were constructed and expressed in Pichia pastoris. METHODS: Using PCR and molecular clone techniques, the fusion genes of IFNα2b-(G4S)n-Tα1 (n=1-3) were constructed and subcloned into the eukaryotic expression vector pPIC9. After transformation of these plasmids into P. pastoris, the expressed fusion proteins IFNα2b-(G4S) n-Tα1 (n=1-3) were obtained. These proteins were purified through diethylaminoethyl (DEAE) affinity chromatography and Superdex 75 gel filtration and analyzed by SDSPAGE and Western blot. Antiviral and E-rosette assays were used to investigate the bioactivities of these fusion proteins. RESULTS: DNA sequencing confirmed that the fusion genes of IFNα2b-(G4S)n-Tα1 (n=1-3) were correctly cloned to the pPIC9 vector. The recombinant IFNα2b-(G4S)n-Tα1 (n=1-3) fusion proteins expressed in P. pastoris were purified with DEAE and Superdex™ 75 gel filtration chromatography. The fusion proteins could be observed on sodium dodecylsulfate-polyacrylamide gel electrophoresis with molecular weight (MW) of 23.2, 22.9, and 22.6 ku, respectively, and reacted to the IFNα2b monoclonal antibody and Tα1 polyclonal antibody. The purified fusion proteins exhibit antiviral activity and can enhance the percentage of E-rosette-forming-cell in E-rosette assay. CONCLUSION: The recombinant IFNα2b-(G4S)n-Tα1 (n=1-3) fusion proteins were successfully expressed in P. pastoris. Purified fusion proteins exhibit both antiviral activity of IFNα2b and immunomodulatory activity of Tα1 in vitro. These results will be the basis for further evaluation of the fusion proteins' function in vivo.