A self-powered time-temperature indicator (TTI) was optimized to enhance the performance of the TTI by modifying a biofuel cell using different immobilization, redox mediators, and status of electrode. The performance of the TTI was measured by output voltage of the TTI. The enzymes and combinations of lacasse mediators (HBT (1-hydroxybenzotriazole), Rupy (Bis-(bipyridine)-(5-aminophenanthroline) ruthenium bis (hexafluorophosphate)), MB (Methylene blue), SDP (4,4- sulfonyldiphenol)) and glucose oxidase mediators (FA (Ferroceneboxaldehyde), DBQ(2,5-dihydroxybenzoquinone), HQS (8-hydroxyquinoline-5sulfonic acid hydrate), FHFP (Ferrocenium hexafluorophosphate)) were immobilized with stabilizers (pyrrole) on a glassy carbon electrode by electrodeposition by applying a square wave, cross-linking and physical immobilization. MWCNTs were used to modify glassy carbon electrodes. MWCNTs coated electrodes produced higher output voltage than uncoated electrodes. The optimum and stable performance of the self-powered TTI was that the output voltage of 64 mV and duration time was 3hr at 25°C, when the combination of Rupy, MB for laccase mediators and HQS, FHFP for glucose oxidase mediators were immobilized on MWCNTs coated electrodes by applying a square wave method. In the application, the concentrations of enzyme and glucose were adjusted to prolong the shelf-life of TTI at much lower output voltage.