The pro-inflammatory cytokine, TNF-α , which plays a major role in the development and persistence of diseases such as Crohn’ s disease, psoriasis, psoriatic arthritis, and rheumatoid arthritis, is the basis for the use of anti-TNF-α therapies. The neutralization of TNF-α or blockage of its binding to the corresponding receptor has mainly served as a therapeutic strategy against some inflammatory diseases. This study aimed to investigate the production of a humanized single chain antibody (scFv) against TNF-α . Therefore, a murine monoclonal antibody, D2 mAb, was selected for humanizing by the complementarity determining region (CDR)-grafting method. Briefly, the replacement of the CDRs from D2 mAb with the specific human single chain scaffold led to the production of a novel humanized single chain fragment variable mAb against human TNF-α (hD2). The subsequent cloning of hD2 into a suitable expression vector, pGEX-6P-1, resulted in the expression of a 52-kDa GST-fusion protein in E. coli, mostly in the form of inclusion bodies. The solubilization and refolding of GSThD2 inclusion bodies was achieved with the addition of 4 M urea and subsequent dialysis to recover the fusion protein in soluble form. Then the soluble GST-hD2 was purified by affinity chromatography through immobilized glutathione. The GST pull-down experiment showed a positive interaction between GST-hD2 and TNF-α protein. Moreover, the results of an MTT assay showed that the purified GST-hD2 has TNF-α neutralizing activity (Kd of 1. 03 nM) and hence hD2 has the potential to be developed into a therapeutic agent. However, more investigation is needed to elucidate the potential of in-vivo TNF-α neutralizing activity of hD2 in comparison to other anti-TNF-α antibodies.