Bifunctional <i>trans</i>-cyclooctene (bTCO) with a carbamate or carbonate at the allylic position and tetrazine provide a promising bioorthogonal click chemistry pair for the click-to-release approach, successfully employed in various biotechnological applications. Herein, we demonstrate a simple and straightforward method to synthesize C<sub>2</sub>TCO, a symmetrical bTCO derivative with two hydroxyl groups at the allylic positions. The efficiently synthesized C<sub>2</sub>TCO at first was selectively functionalized with a fluorophore (C<sub>2</sub>TCO-FL), and the conjugate was labeled onto monoclonal antibodies (Ab-C<sub>2</sub>TCO-FL). The fluorophore of Ab-C<sub>2</sub>TCO-FL was easily removed from the antibody through the mild treatment of tetrazine, enabling multicycle fluorescent bioimaging. Next, an antibody-drug conjugate targeting PD-L1 was prepared using the linker based on C<sub>2</sub>TCO. The cytotoxic payload was efficiently released from the antibody upon tetrazine treatment, which induced cellular cytotoxicity.