<p>Osteosarcoma is a highly malignant tumor with poor prognosis. Current CAR-T cell therapies for osteosarcoma are predominantly designed with single targets, but their efficacy remains unsatisfactory. In this study, a novel bispecific CAR-T cell was developed to provide an experimental basis for improving the therapeutic outcome of osteosarcoma. Single-cell RNA sequencing (scRNA-seq) identified two antigens highly expressed in osteosarcoma cells, ANXA2 and CD147, whose expression was further validated at the tissue level by qRT-PCR, flow cytometry, and immunohistochemistry. Based on a second-generation CAR backbone, a bispecific ANXA2/CD147 CAR-T construct was generated using magnetic bead sorting, primary T-cell culture, and lentiviral transduction, achieving a transduction efficiency of 47.1%. LDH release assays demonstrated that bispecific CAR-T cells exhibited significantly greater cytotoxicity against tumor cells than single-target and control groups. ELISA confirmed that bispecific CAR-T cells released higher levels of effector molecules, including GZMB and TNFα. In a subcutaneous CDX model, bispecific CAR-T cells displayed superior antitumor activity and greater T-cell infiltration. In a paw pad xenograft model, mice treated with bispecific CAR-T cells exhibited the smallest tumor volumes, lowest tumor weights, and reduced rates of lymph node metastasis. Furthermore, PDX models confirmed that bispecific CAR-T cells effectively suppressed osteosarcoma growth. ScRNA-seq of tumors derived from CDX models and immunohistochemistry revealed markedly increased infiltration of M1 macrophages in the bispecific group. Collectively, this study successfully generated a bispecific ANXA2/CD147 CAR-T cell with robust antitumor activity, providing a promising new strategy for the immunotherapy of osteosarcoma.</p>