Using phage display technology (PDT), we have recently isolated single-chain variable fragment (scFv) antibodies (Ab) against some pivotal molecular markers involved in malignancies and systemic inflammation including human tumor necrosis factor-alpha (TNF-α, GI:367465798). Downstream purification and characterization of scFv antibodies is a challenging issue, which may impose substantial impacts on quality of the final product(s). Of various purification methods, affinity chromatography has widely been used in the pharmaceutical grade downstream processing (PGDP) of proteins (e.g., monoclonal antibody (mAb) and scFvs). To pursue an optimized PGDP, in the current study, we have capitalized PDT for upstream selection of anti-TNF-α scFvs and protein A affinity chromatography (PAAC) for downstream extraction and purification of the scFvs from the crude medium secreted and periplasmic fractions of HB2151 cells. The versatility of the PDT selection was validated using SDS-PAGE electrophoresis, western blot, dot blot, ELISA and fluorescence microscopy. SDS-PAGE western blot analyses displayed a 17 kDa scFv with high purity (> 98%), while dot blot and ELISA analyses showed high specificity and binding affinity of the purified scFv antibody fragments toward human TNF-α at nM range. Fluorescence microscopy further confirmed detection of TNF-α in Raji B lymphoblast cells. Finally, based on our findings, we believe that these PDT selected and PAAC processed scFvs may be used as targeting and/or therapy agent for TNF-α mediated diseases. Further, to increase the production yield, downstream purification techniques need to be optimized for the large scale production of scFv antibodies.