Objective: New Strategy for solubilization and refolding of recombinant human Interferon α2b inclusion bodies was established to obtain a high solubilization and refolding rate of recombinant human Interferon α2b inclusion bodies from E. coli Gene Overexpression System.Method: The IFN-α2b inclusion bodies were solubilized with solubilizing buffer of 2mol/L urea. Refolding was performed via two steps, diluting by pulse adding the solubilized IFN α2b sample and dialysing it slowly using ultrafiltration 5K step by step. Finally refolded IFN α2b was purified through Cu 2+ chelate affinity chromatography. Results: The urea concentration of 2mol/L in the solubilizing buffer gave the solubilization rate of 89.73%. Diluting the solubilized IFN α2b sample by pulse adding gave the refolding rate of 78.53%. The multi steps dialysis through ultrafiltration membrane 5K gave the antiviral activity recovery of 98.73%. The purification through one step Cu 2+ chelate affinity chromatography raised the specific activity of IFN sample to 1.4×10 8 U/mg protein and 12% SDS-PAGE showed single band of purified IFN-α2b at expected MW height, whose purity was 99.8%. Conclusion: Large-scale production of recombinant human Interferon α2b (IFN-α2b) in E. coli with a thermoinducible overexpression system was established by applying an effective solubilizing and refolding processes of interferon α2b inclusion bodies (IBs). Solubilization and refolding rate of IFN-α2b IBs were 89.71% and 79.87% respectively. Refolded IFN-α2b was purified through one-step immobilized metal affinity chromatography to give a pure bioactive IFN-α2b with the specific activity of 1.4×10 8 IU/mg protein and with the recovery rate over 52.89%.