A new technology for renaturation with simultaneous purification of the recombinant human interferon- (rhIFN-) in downstream of biotechnology is presented. The strategies to develop the new technology in industry scale were suggested. Based on chemical equilibrium and molecular interactions, the principle of rhIFN-refolding by HPHIC was described. The kind of stationary and mobile phases were evaluated and found the former to contribute to the rhIFN-refolding more than the latter. The extract containing the rhIFN-in gram scale in 7.0mol L−1 guanidine hydrochloride solution of 700mL was directly pumped into a unit of simultaneous renaturation and purification of proteins (USRPP, 10×300mmi.d.) packed by small particle packings of hydrophobic interaction chromatography and a satisfactory recovery of bioactivity and mass of the rhIFN-was obtained. With flow rate 100mLmin−1 and a gradient elution by only one step in 4h, the purity and specific bioactivity approach to 95% and 8.7×107 IU−1mg, respectively. To evaluate the goodness of the presented new technology in this study, a usual method with the renaturation by dilution method firstly and then purification with a series of LC in literature was employed to compare with each other. The obtained result in terms of purity, recoveries of mass and bioactivity, cost time as well as expenses, the former is much better than the latter. Comparing the total bioactivity of rhIFN- in the extract before to that after the renaturation by the USRPP, the total bioactivity of rhIFN- increased 62-fold.