A sensitive sensor for the determination of hydrogen peroxide (H₂O₂) was fabricated by immobilizing cytochrome c (cyt c) on binary self-assembled monolayers (SAMs) of 5-(1,2-dithiolan-3-yl)valeric acid (thioctic acid, T-COOH) and 5-(1,2-dithiolan-3-yl)valeramide (thioctic acid amide, T-NH₂) on gold electrodes. The electrochemical characteristics of immobilized cyt c were investigated by cyclic voltammetry, electrochemical impedance spectroscopy and chronoamperometry. The modified electrode showed good electrocatalytic activity towards the reduction of H₂O₂. The catalytic current is linear with the H₂O₂ concentration ranging from 10.0 to 300 µM with a correlation coefficient of 0.9996. The apparent Michaelis-Menten constant (K_m^app) was found to be 619 µM. The sensor achieved 95% of the steady-state current within 10 s, exhibiting high sensitivity (1.7 nA µM^-1 cm^-2).