Objective To construct lentiviral vectors for the expression of monovalent antibody against human c-mesenchymal epithelial transition factor (c-Met) using anti-c-Met chimeric antibody ch3E1D7 plasmid, and test the affinity and neutralizing ability of the purified monovalent antibody in transfected HEK293T cells. Methods The anti-c-Met monovalent antibody was designed, namely mono3E1D7. Three different lentiviral expression vectors of the monovalent antibody were then constructed using genetic engineering technology. The three expression vectors were co-transfected in HEK293T cells to express the monovalent antibody, which was later purified by protein A-sepharose 4B affinity chromatography. The antibody structural integrity was identified by SDS-PAGE. Ability of the monovalent antibody to bind and neutralize hepatocyte growth factor (HGF) was tested by ELISA. Results Heavy, light and Knob chains of the mono3E1D7, with molecular masses of about 55, 25 and 30 kD, respectively, were observed on reduced 10% SDS-PAGE. ELISA showed that the expressed protein could bind to c-Met specifically and neutralize c-Met/HGF binding. Conclusion Monovalent antibody targeting c-Met has been successfully constructed, expressed and identified, which could help to study the important role of monovalent antibody targeting to c-Met in following experiments.