Trophoblast cell surface antigen 2 (TROP2) has become one of the effective therapeutic targets by antibody-drug conjugates (ADCs) such as sacituzumab govitecan which US FDA approved in 2020. TROP2 is reported to be overexpressed, and involved in tumor cell proliferation, invasion, metastasis, and poor prognosis in several types of solid tumor. Several clinical trials of anti-TROP2 ADCs are ongoing worldwide in patients with breast and lung cancer. Here, using a Cell-Based Immunization and Screening (CBIS) method, we developed a novel anti-TROP2 monoclonal antibody (clone TrMab-6; mouse IgG_2b, kappa). TrMab-6 was found to be applicable for many experiments, including flow cytometry, Western blotting, and immunohistochemistry. Furthermore, we investigated the potential of TrMab-6 for in vitro antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) activities, and in vivo antitumor activities. TrMab-6 strongly induced anti-tumor effects against breast cancer cell lines, including MCF7, MDA-MB-231, and MDA-MB-468 in vitro. In vivo experiments revealed that TrMab-6 significantly reduced tumor growth on breast cancer xenograft models. These results indicated that TrMab-6 could be a promising treatment option for TROP2-expressing breast cancers.