This study aimed to obtain recombinant fusion protein of thymosin alpha1(TM-a1) and consensus IFNa (IFNa-con) which have bath TM-a1 and IFNα-con activities. The DNA sequence for the fusion protein was cloned into expression vector of pET-22b (+) and expressed in BL21 (DE3)-Codon plus-RP-X. The expressed product (TM-a1-IFN-con) was soluble, and amounted to more than 20% in total proteins of E. coli. By precipitation of (NH4)2SO4, hydrophobic interaction chromatography (HIC, Phenyl Sepharose 6 Fast Flow), anion-exchange chromatography (Q Sepharose Fast Flow), cation-exchange chromatography (SP Sepharose Fast Flow) and gel filtration (Sephadex G-75), it was purified to more than 96% purity. The activity of fusion protein for antivirus was tested by cytopathic-effect inhibition assay and activity for promoting lymphocyte proliferation is tested by cell proliferative assay. The activity for antivirus was higher than commercial IFNα1b and IFNα2a and activity for promoting lymphocyte proliferation was similar to commercial TM-α1. The fusion protein had better effect for anti-HBV in vitro, its effect was stronger than combination of IFNa and TM-a1 and cell toxicity was less than combination of IFNa and TM-a1. The above results show that it has effect bath antivirus of IFNa and promoting lymphocyte proliferation of the soluble fusion protein expressed in E. coli.