The combination of porous carbon nanofiber (PCNF) and room-temperature ionic liquid (RTIL) provided a suitable microenvironment for heme-proteins to transfer electron directly. Hemoglobin, myoglobin, and cytochrome c incorporated in PCNF/RTIL films exhibited a pair of well-defined, quasi-reversible cyclic voltammetric peaks at about -0.28 V vs. SCE in pH 7.0 buffers, respectively, characteristic of the protein heme Fe(III)/Fe(II) redox couples. The cyclic voltammetry and electrochemical impedance spectroscopy were used to characterize the modified electrode. The heme/PCNF/RTIL/CHIT films were also characterized by UV-vis spectroscopy, indicating that heme-proteins in the composite film could retain its native structure. Oxygen, hydrogen peroxide, and nitrite were catalytically reduced at the heme/PCNF/RTIL/CHIT film modified electrodes, showing the potential applicability of the films as the new type of biosensors or bioreactors based on direct electrochemistry of the redox proteins. (C) 2010 Elsevier Ltd. All rights reserved.