Abstract: In this study, a synthetic gene encoding 166 residues of interferon-β was constructed. For an efficient expression of IFN- β in E. coli, the synthetic gene was designed according to the E. coli codon usage. Cysteine 17 was replaced with serine to avoid multimer formation and random intramolecular disulfide bridges. The sequence of the designed gene was analyzed with Web Cutter and RNA secondary structure softwares to make sure about the cloning and expression of the recombinant gene in the proposed cloning and expression vectors. Twelve overlapping oligonucleotides ranging from 55 to 83 in length were designed. A step-wise assembly polymerase chain reaction was used for the construction of the synthetic gene through three steps PCR programs using overlapping adjacent primers as well as PCR products of a previous steps. The final PCR product was further amplified and cloned into T/A cloning vector. The constructed gene was sub-cloned into pQE30, pKK322 and pGEMEX-1 expression vectors and the expression was analyzed with Western-blotting.