Alloxan is a cytotoxic organic molecule that selectively damages islet β-cell in the pancreas and disrupts the production of insulin, causing alloxan-induced diabetes. Herein, for the first time, we explored alloxan molecule as a new co-reactant species for the standard ECL luminophore [Ru(bpy)3]2+in both physiological and alkaline pH medium. The simultaneous CV and ECL studies confirm that the reversible redox peak of [Ru(bpy)3]2+ becomes in irreversible nature at + 1.15 V (vs. Ag/AgCl) and its corresponding ECL peak also consistently increases with each addition of alloxan molecule. The CV, UV-Visible, Raman, NMR and HPLC results confirm that at pH 7.4 and above, the alloxan molecule is chemically transformed into alloxanic acid. The electron spin resonance (ESR) spectra further endorse that the oxidation peak at + 1.0 V is due to oxidation of alloxanic acid into alloxanic radical, which ultimately serves as anodic co-reactant for [Ru(bpy)3]2+. The proposed new ECL oxidative-reduction mechanism via energetic electron transfer takes place between alloxanic radical and the electro-oxidized [Ru(bpy)3]3+. The developed an ECL based sensor which shows more sensitive and selective detection of alloxan with a linear ranges of 100 nM - 2 mM at pH 7.4 and 10 nM - 2 mM at pH 11 with a limit of detection (LOD) value of 0.97 nM & 0.70 nM (S/N = 3) respectively. Moreover, the developed ECL based sensor is successfully applied to the sensing of alloxan in the real samples that proves the viability of the adopted ECL sensor methodology is suitable for detecting the alloxan molecule in food as well as clinical samples.