Enzymatic biofuel cells (EBCs) using non-toxic and naturally derived flavonoid biomediators as anodic catalysts are introduced. In this study, hesperidin, a flavonoid extracted from citrus fruits, served as a biomediator with flavine adenine dinucleotide dependent glucose dehydrogenase (FADGDH). Hesperidin exhibits several advantages, including a low onset potential, a robust catalytic activity, and an excellent stability for glucose oxidation reaction (GOR). This superiority can be attributed to its unique chemical structure and electrochemically activated capability. For its activation, a catechol group was produced on the right side of the phenyl ring of hesperidin, existing in both chemically and physically adsorbed forms. Following activation, a redox reaction occurred at a buckypaper (BP) supported electrode (BP/Activated Hesperidin (A-Hesperidin)) at −0.05 and 0.15 V (vs. Ag/AgCl). When BP/A-Hesperidin/FADGDH was employed, the reactivity of GOR reached 0.61 mA cm−2 at 0.3 V with 10 mmol L−1 glucose. These performances surpass those reported in similar research studies. When the EBC using BP/A-Hesperidin/FADGDH was operated, its maximum power density (MPD) reached 212.84 ± 17.97 μW cm−2 with 10 mmol L−1 glucose under air conditions. This confirms that A-Hesperidin can induce higher EBC performances than conventionally used mediators, which are often toxic and expensive. Display omitted