Erythropoietin-producing hepatocellular receptor B6 (EphB6) is a member of the largest Eph subfamily of receptor tyrosine kinases. EphB6 is widely expressed in various tissues and regulates cellular homeostasis by interacting with its membrane-bound ephrin ligands and other receptors. EphB6 is involved in cancer pathology despite lacking kinase activity. Therefore, developing sensitive monoclonal antibodies (mAbs) for EphB6 has been desired for treatment, diagnosis, and further analysis of EphB6. This study established a novel specific and sensitive anti-human EphB6 mAb clone Eb6Mab-3 (mouse IgG1, kappa) by the Cell-Based Immunization and Screening (CBIS) method. In flow cytometry, Eb6Mab-3 demonstrated reactivity with EphB6-overexpressed Chinese hamster ovary-K1 cells (CHO/EphB6) and endogenously EphB6-expressing DLD-1 colorectal cancer cells. Cross-reactivity of Eb6Mab-3 was not observed. Eb6Mab-3 demonstrated a moderate binding affinity (dissociation constant; KD) for CHO/EphB6 (KD: 3.8×10-8 M) and a high binding affinity for DLD-1 (KD: 1.9×10-9 M). Eb6Mab-3 can detect EphB6 protein in CHO/EphB6 lysate in western blot. Therefore, Eb6Mab-3, established by the CBIS method, could be valuable for analyzing the EphB6-associated cellular functions and has potential applications in diagnosis and treatment with specificity and high affinity for cancer cells.