This paper reports on the fabrication and characterization of hemoglobin (Hb)-colloidal silver nanoparticles (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 1.83s −1 and displayed the same features of a peroxidase in the electrocatalytic reduction of oxygen and hydrogen peroxide (H 2 O 2 ). The linear range for the determination of H 2 O 2 was from 0.75μM to 0.216mM with a detection limit of 0.5μM (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.