Three-dimensionally (3D) ordered macroporous active carbon has been fabricated and used as electrode substrate for the direct electrochemistry of horse heart cytochrome c (Cyt c). The Cyt c immobilized on the surface of the ordered macroporous active carbon shows a pair of well-defined and nearly reversible redox waves at the formal potential of −0.033 V in pH 6.8 phosphate buffer solution. The interaction between Cyt c and the 3D macroporous active carbon makes the formal potential shift negatively compared to that of Cyt c in solution. Spectrophotometric and electrochemical methods have been used to investigate the interaction between Cyt c and the porous active carbon. The immobilized Cyt c maintains its biological activity, and shows a surface controlled electrode process with the electron-transfer rate constant ( k s) of 17.6 s −1 and the charge-transfer coefficient ( a) of 0.52, and displays the features of a peroxidase in the electrocatalytic reduction of hydrogen peroxide (H 2O 2). A potential application of the Cyt c-immobilized porous carbon electrode as a biosensor to monitor H 2O 2 has been investigated. The steady-state current response increases linearly with H 2O 2 concentration from 2.0 × 10 −5 to 2.4 × 10 −4 mol l −1. The detection limit (3 σ) for determination of H 2O 2 has been found to be 1.46 × 10 −5 mol l −1.