Tetracycline analogues (oxytetracycline, doxycycline, and tetracycline) can inactivate copper, zinc superoxide dismutase (CuZnSOD). The interactions between tetracycline analogues and CuZnSOD were studied by using the UV– visible absorption and spectrofluorimetric method. Tetracycline analogues can cause fluorescence quenching of CuZnSOD by a static mechanism. The absorption spectrum and quenching constant all support this conclusion. The binding constants of tetracycline analogues with CuZnSOD were obtained at various temperatures. Based on the Fo¨rster nonradioactive energy transfer theory, we obtain the distance between the donors and acceptors. Based on the thermodynamic parameter we also determined that the main force acting between them is electrostatic gravitation. Tetracycline analogues can decrease the activity of CuZnSOD purified from garlic and the whole blood from rabbits injected with tetracycline. Such results provide a reference for clinical diagnosis using the activity level of CuZnSOD, which varies in patients with different diseases.