The treatment of breast cancer (BC) remains a formidable challenge due to the emergence of drug resistance, necessitating the exploration of innovative strategies. Chimeric antigen receptor (CAR)-T cell therapy, a groundbreaking approach in hematologic malignancies, is actively under investigation for its potential application in solid tumors, including BC. Trophoblast cell surface antigen 2 (Trop2) has emerged as a promising immunotherapeutic target in various cancers and is notably overexpressed in BC. To enhance therapeutic efficacy in BC, a fourth-generation CAR (CAR4) construct was developed. This CAR4 design incorporates an anti-Trop2 single-chain variable fragment (scFv) fused with three costimulatory domains -CD28/4-1BB/CD27, and CD3ζ. Comparative analysis with the conventional second-generation CAR (CAR2; 28ζ) revealed that anti-Trop2 CAR4 T cells exhibited heightened cytotoxicity and interferon-gamma (IFN-γ) production against Trop2-expressing MCF-7 cells. Notably, anti-Trop2 CAR4-T cells demonstrated superior long-term cytotoxic functionality and proliferative capacity. Crucially, anti-Trop2 CAR4-T cells displayed specific cytotoxicity against Trop2-positive BC cells (MDA-MB-231, HCC70, and MCF-7) in both two-dimensional (2D) and three-dimensional (3D) culture systems. Following antigen-specific killing, these cells markedly secreted interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-α), IFN-γ, and Granzyme B compared to non-transduced T cells. This study highlights the therapeutic potential of anti-Trop2 CAR4-T cells in adoptive T cell therapy for BC, offering significant promise for the advancement of BC treatment strategies.