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Recombinant human interferon α-2b is an FDA-approved drug for monotherapy or in combination therapy with other drugs for hepatitis and cancers. It belongs to a family of homologous proteins involved in antiviral, antiproliferative, and immunoregulatory processes. A different expression system has been used for overexpression of this protein. Escherichia coli expression system is a highly characterized host and various expression settings have been developed based on its properties. However, finding the best conditions for the overexpression of recombinant human interferon α-2b remains to be addressed. In this study, the expression of synthetic human interferon α-2b gene in Escherichia coli was greatly improved by adjusting the expression condition. In this regard, a recombinant gene was designed and codon optimized for the periplasmic expression of this protein. Then, gene subcloning was employed to insert the synthesized gene into the pET22b expression vector. Thereafter, the response surface methodology was employed to design 20 experiments to find out the optimum points for isopropyl β-D-1-thiogalactopyranoside concentration, post-induction period, and the cell density of induction (OD600). The expression fluctuations were assessed by using the real-time polymerase chain reaction method. Our results indicated that the synthetic human interferon α-2b gene was successfully codon optimized and subcloned into the expression vector. The realtime polymerase chain reaction results revealed that the optimum levels of the selected parameters are 0.27 mM for isopropyl β-D-1-thiogalactopyranoside concentration, 7.98 H for the post-induction period, and 3.93 for cell density (OD600). These optimized conditions led to a 3.5-fold increase in the rhIFNα2b expression, which is highly promising for large scale rhIFNα2b overexpression. [ABSTRACT FROM AUTHOR]