A simple and sensitive electrochemical sensor based on nickel oxide nanoparticles/riboflavin-modified glassy carbon (NiONPs/RF/GC) electrode was constructed and utilized to determine H2O2. By immersing the NiONPs/ GC-modified electrode into riboflavin (RF) solution for a short period of time (5–300 s), a thin film of the proposed molecule was immobilized onto the electrode surface. The modified electrode showed stable and a well-defined redox couples at a wide pH range (2–10), with surface-confined characteristics. Experimental results revealed that RF was adsorbed on the surface of NiONPs, and in comparison with usual methods for the immobilization of RF, such as electropolymerization, the electrochemical reversibility and stability of this modified electrode has been improved. The surface coverage and heterogeneous electron transfer rate constants (ks) of RF immobilized on a NiOx–GC electrode were approximately 4.83 9 10-11 mol cm-2, 54 s-1, respectively. The sensor exhibits a powerful electrocatalytic activity for the reduction of H2O2. The detection limit, sensitivity and catalytic rate constant (kcat) of the modified electrode toward H2O2 were 85 nM, 24 nA lM-1 and 7.3 (±0.2) 9 103 M-1 s-1, respectively, at linear concentration rang up to 3.0 mM. The reproducibility of the sensor was investigated in 10 lM H2O2 by amperometry, the value obtained being 2.5 % (n = 10). Furthermore, the fabricated H2O2 chemical sensor exhibited an excellent stability, remarkable catalytic activity and reproducibility.