C-C motif chemokine receptor-8 (CCR8) belongs to class A of G protein-coupled receptors. CCR8 interacts with the specific chemokine ligand CCL1/I-309 in humans, which is produced by various cells, including tumor-associated macrophages and regulatory T cells (Treg). CCR8 is highly expressed on Treg and T-helper 2 cells recruited to the inflammation site and is implicated in allergy, asthma, and cancer progression. CCR8+Treg cells have been suggested an important regulator in the immunosuppressive tumor microenvironment. Therefore, it has been proposed for use in the development of sensitive monoclonal antibodies targeting CCR8. This study developed a specific mAb for human CCR8 (hCCR8), which is useful for flow cytometry by employing the Cell-Based Immunization and Screening (CBIS) method. The established anti-hCCR8 mAb (C₈Mab-21; mouse IgM, kappa) demonstrated reactivity with hCCR8-overexpressed Chinese hamster ovary-K1 (CHO/hCCR8) cells, TALL-1 (human adult acute T-lymphoblastic leukemia), CCRF-HSB2 (human T-lymphoblastic leukemia), and natural killer cells expressing endogenous hCCR8, as confirmed by flow cytometry. Furthermore, EC₅₀ values of C8Mab-21 for CHO/hCCR8 and TALL-1 were determined as 6.5 × 10⁻⁸ M and 2.0 × 10⁻⁸ M, respectively. C₈Mab-21, established by the CBIS method, provides a useful tool for analyzing the hCCR8-related biological response using flow cytometry.