The development of an amperometric sensor utilizing tetrathiafulvalene (TTF) as an electron shuttle between immobilized lactate oxidase and a glassy carbon electrode is described. The problem of mediator leaking in electrodes using TTF is overcome by employing Nafion membrane that prevents leaching of positive TTF+ ions by electrostatic attraction. Stability, interference, and the factors such as oxygen, applied potential, and pH influencing the performance of the lactate sensor are examined and discussed. The response of the sensor to lactate under N-2 saturation reaches 95% steady-state current within 40s.