Recombinant expression and EGFR-binding activity assay of single chain nimotuzumab (nimotuzumab scFv) is reported in this study. The scFv was produced in VH-linker-VL format in Origami™ 2(DE3)pLysS bacterial cells and purified using Ni-TNA resin column. 3-D structure prediction using I-TASSER (Iterative Threading Assembly Refinement) server and analyzing the predicted models using YASARA (Yet Another Scientific Artificial Reality Application) viewer revealed that VH and VL domains assemble into a correctly-folded single chain antibody that is able to bind EGFR. The scFv was evaluated in ELISA and western blot tests and proven to be able to bind EGFR-overexpressing cancer cells (A-431 cells in an efficient manner but unable to recognize cancer cells expressing low levels of EGFR (MCF-7 breast cancer cells).