The use of heat treated carbon nanofibers, known as carbon nanochips (CNCs) for the studies of the direct electrochemistry and electrocatalytic properties of heme proteins, is demonstrated. A glassy carbon electrode (GCE) was modified with CNCs, and hemoglobin (Hb) was immobilized on the modified electrode surface by casting a film of Hb. Nafion was employed to fix the CNCs and Hb tightly on the Surface of the GCE. The modified electrode was characterized by scanning electron microscopy. Ultraviolet-visible and Fourier transform infrared spectroscopy showed that Hb immobilized in the CNC film remained in its native structure. Electrochemical impedance spectroscopy and cyclic voltammetry (CV) were employed for electrochemical studies. The results showed that the presence of CNCs in the film can greatly enhance the electrochemical response of Hb. A pair of well-defined reversible CV peaks was observed, and the formal potential of the heme Fe(III)/Fe(II) redox couple was found to be -253 mV [vs Ag/AgCl (saturated KCl)]. The apparent heterogeneous electron-transfer rate constant (k(s)) was estimated as 2.54 s(-1). The modified electrode showed excellent electrocatalytic behavior to hydrogen peroxide (H2O2), trichloroacetic acid, and sodium nitrite. H2O2 had a linear current response from 0.5 to 30 mu M (R-2 = 0.9997; n = 5) with a detection limit of 0.05 mu M when the signal-to-noise ratio was 3 and the apparent Michaelis-Menten constant (K-m(app)) was 21.55 mu M. These values suggest that CNCs are the best matrix described so far for the development of biosensors, far superior to untreated carbon nanofibers. The direct immobilization of proteins onto the Surface of CNCs is shown to be a highly efficient method for the development of a new class of very sensitive, stable, and reproducible electrochemical biosensors.