Epidermal growth factor receptor (EGFR) is an attractive target for the treatment of epithelial cancers. However, EGFR antagonists have low clinical response rates and frequently induce resistance mainly caused by the hypermutation of the extracellular and intracellular domains and the heterodimerization of EGFR. Dimerization plays a key role in the activation of the EGFR family of receptors. Thus, targeting the highly conserved dimer interface of EGFR maybe an attractive strategy for improving the clinical response of anti-EGFR therapies. In this work, we report a monoclonal antibody (mAb) called Antidimer 5G9 that targets the β-hairpin within the dimer interface of EGFR, which is 100% homologous with mouse EGFR. Antidimer 5G9 belongs to the isotype IgG1 and bound with an affinity constant of 1.837×10L/mol. Just as designed and expected, Antidimer 5G9 could recognize and bind to human EGFRs or mouse EGFRs on the surface of human breast cancer cells of the MDA-MB-231 cell line or mouse Lewis lung cancer cells (LLC), effectively inhibiting growth and inducing apoptosis in the targeted cells. This work lays the foundation for further studies investigating the feasibility of this strategy for targeting the dimer interface of EGFR and developing novel anti-EGFR drugs.