Claudin 6 (CLDN6) is a tight junction transmembrane protein. It is hardly detected in adult normal tissues, while aberrantly expressed in a variety of tumor types, making it an ideal target for cancer treatment. As a co-stimulatory receptor, 4-1BB elicits powerful activation signal upon ligation by agonist antibody, which is regarded as an effective solution for improving immune checkpoint inhibitors response and overcoming resistance. However, 4-1BB agonist antibody development is usually halted by systemic safety problems, especially liver toxicity. Here, we generated a CLDN6-4-1BB bispecific antibody TJ-C64B, which could conditionally activate T cells through 4-1BB stimulation upon CLDN6 engagement. Besides, the competent Fc portion granted TJ-C64B the capability of specifically depleting CLDN6-expressing tumor cells and Tregs in tumor microenvironment reported to be highly expressed 4-1BB. The unique properties of TJ-C64B positions it as a potential novel immunotherapy for ovarian cancer and other CLDN6 positive tumors.