A novel nafion-riboflavin membrane was constructed and characterized by the scanning electron microscopy (SEM), transmission electron microscopy (TEM), UV-visible spectroscopy and cyclic voltammetric techniques. The estimated average diameter of the designed nanoparticles was about 60 nm. The functional membrane showed a quasi-reversible electrochemical behaviour with a formal potential of -562 ± 5 mV (vs Ag/AgCl) on the gold electrode. Some electrochemical parameters were estimated, indicating that the system has good and stable electron transfer properties. Moreover, horseradish peroxidase (HRP) was immobilized on the riboflavin-nafion functional membrane. The electrochemical behaviour of HRP was quasi-reversible with a formal potential of 80 ± 5 mV (vs Ag/AgCl). The HRP in the film exhibited good catalytic activity towards the reduction of H2O2. It shows a linear dependence of its cathodic peak current on the concentration of H2O2, ranging from 10 to 300 μM.