This paper reports on the fabrication and characterization of hemoglobin (Hb)-colloidal silver nanopartides (CSNs)-chitosan film on the glassy carbon electrode and its application on electrochemical biosensing CSNs could greatly enhance the electron transfer reactivity of Hb as a bridge In the phosphate buffer solution with pH value of 7 0 Hb showed a pair of well-defined redox peaks with the formal potential (E-0) of -0 325V (vs SCE) The immobilized Hb in the film maintained its biological activity showing a surface-controlled process with the heterogeneous electron transfer rate constant (k(s)) of 1 83 s(-1) and displayed the same features of a peroxidase in the electrocatalytic reduction of oxygen and hydrogen peroxide (H2O2) The linear range for the determination of H2O2 was from 0 75 mu M to 0 216 mM with a detection limit of 0 5 mu M (S/N = 3) Such a simple assemble method could offer a promising platform for further study on the direct electrochemistry of other redox proteins and the development of the third-generation electrochemical biosensors (C) 2010 Elsevier Ltd All rights reserved