A biofunctional hybrid composite was constructed by assembling hydroxyapatite (HA) onto carboxylic group-functionalized carbon nanofibers (FCNFs). The FCNFs was obtained from acid treatment of carbon nanofibers (CNFs) which were synthesized by the combination of electrospinning and thermal treatment processes. The obtained carbon nanofibers-hydroxyapatite composite (FCNFs-HA) was characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD) and FTIR spectroscopy. Cytochrome c (Cyt c) was successfully immobilized in this three-dimensional FCNFs-HA composite and the electron transfer rate constant (k(s)) was evaluated to be 3.66 s(-1) according to Laviron's equation. And the surface coverage (Gamma*) was estimated to be 8.1 x 10(-10) mol cm(-2). Cyt c immobilized in FCNFs-HA composite exhibited a good electrocatalytic activity towards the reduction of hydrogen peroxide (H(2)O(2)). The catalytic current is linear to the H(2)O(2) concentration in the range of 2.0 mu M to 8.7 mM (r=0.9996: n = 28), and the detection limit was 0.3 mu M based on the criterion of a signal-to-noise ratio of 3. (C) 2011 Elsevier B.V. All rights reserved.