Abstract: The aim of this study was to clone and optimize production of a recombinant antibody against human leptin receptor which showed inhibitory effects on leptin signalling. DNA sequence encoding Fab fragment of a mouse monoclonal antibody was cloned into pComb3 vector. E. coli was transformed with the plasmid and recombinant antibody was produced. In order to achieve high level of protein expression, recombinant antibody production was performed using different E. coli strains and culture conditions. Antibody production was detected using ELISA (Enzyme-linked immunosorbent assay), Western blotting and Dot blotting techniques. Recombinant Fab fragment of antibody was produced in E. coli successfully at reasonable quantities with comparable affinity to the antigen compared to antibody produced by original hybridoma cells. The results showed that among different culture media, SB medium was the best medium and among different temperatures 22°C was the best temperature for recombinant antibody production. From three different E. coli strains (JM109. XL1-Blue and BL21) tested, JM109 produced the highest levels and among different IPTG concentrations 1 mM induced the highest level of antibody production.