tA simple and sensitive electrochemical sensor based on nano-ruthenium oxide/riboflavin modified glassycarbon (GC/RuOx/RF) electrode was constructed and utilized to determine H2O2. By immersing theGC/RuOx modified electrode into RF solution for a short period of time (5–70 s), a thin film of the proposedmolecule was immobilized onto the electrode surface. The modified electrode showed stable and a well-defined redox couples at a wide pH range (1–10), with surface confined characteristics. Experimentalresults revealed that RF was adsorbed on the surface of RuOx nanoparticles (NPs) and in comparison tousual methods for the immobilization of RF, such as electropolymerization, the electrochemical reversibil-ity and stability of this modified electrode has been improved. The heterogeneous electron transfer rateconstant (ks) and the surface coverage of immobilized RF on the GC/RuOx electrode was obtained as16.8 s−1and 2.2 × 10−11mol cm−2, respectively. The electrocatalytic activity of the modified electrodetoward hydrogen peroxide reduction was investigated and it was found that the GC/RuOx/RF electrodeillustrates electrocatalytic activity toward H2O2reduction at reduced overpotential. The catalytic rateconstant (kcat) of the modified electrode toward H2O2is 9.1 × 103M−1s−1. This biosensor shows excel-lent sensitivity toward H2O2in buffer solution (pH 7) with a wide linear range from 0.15 M to 3 mM.The detection limit and sensitivity are 150 nM and 35 nA M−1respectively. Furthermore, the fabricatedH2O2chemical sensor exhibited excellent stability, remarkable catalytic activity and reproducibility.