Interferon-kappa (IFN-κ) has important biofunctions such as antivirus, antitumor, and immunomodulation. The unique secretion and physiological characteristics make it a promising medicine in clinical treatments. The recombinant human interferon-kappa (rhIFN-κ) expressed as inclusion bodies in E. coli require in vitro refolding to restore its biological functions, yet it tends to form precipitates in the process. Moreover, IFN-κ contains two disulfide bonds, but they are difficult to be correctly oxidized during the normal refolding procedure. In this work, a strong anionic surfactant, sodium dodecyl sulfate (SDS), was used to solubilize the inclusion bodies, and polyol 2-methyl-2,4-pentanediol (MPD) was added to the refolding buffer to gradually strip the SDS from the protein during the renaturation process to complete the refolding of rhIFN-κ. Experimentation showed that identified by the oxidative degree of its two disulfide bonds, there were mainly three species of rhIFN-κ after refolding: the completely oxidated rhIFN-κ, partially oxidated intermediate, and unoxidized species. For the SDS/MPD refolding system, the refolding yield for the correctly oxidated rhIFN-κ was closely related to the concentration and ratio of SDS and MPD in the renaturation system. Insufficient SDS or excessive MPD might result in forming aggregates or precipitates, while the correct oxidization of the disulfide bond would be suppressed at high SDS/MPD concentration ratios. Upon further optimization of the redox system and the solution pH, the optimal buffer was found by adding 0.05wt% SDS, 1 mol/L MPD, 0.2 mmol/L GSSG, and 0.1 mmol/L GSH to 20 mmol/L Tris at pH=9.5. Moreover, increasing protein concentration to 2.0 mg/mL would not significantly decrease the refolding yield. Under the optimized condition, the refolding yield could reach 66% after incubation for 24 hours at room temperature. Further purification with reversed-phase liquid chromatography could effectively remove the misfolded species as well as other impurities, achieving purity of 90% rhIFN-κ with only one band in non-reducing SDS-PAGE. The results showed that the SDS/MPD system could effectively suppress aggregation while increasing the rate of correct oxidization of the disulfide bonds. Our study lay a solid foundation for the production and clinical applications for rhIFN-κ in the future.