Thermally two-dimensional lattice graphene (GR) and biocompatibility chitosan (CS) act as a suitable support for the deposition of palladium nanoparticles (PdNPs). A novel hydrogen peroxide (H2O2) biosensor based on immobilization of hemoglobin (Hb) in thin film of CS containing GR and PdNPs was developed. The surface morphologies of a set of representative membranes were characterized by means of scanning electron microscopy and showed that the PdNPs are of a sphere shape and an average diameter of 50 nm. Under the optimal conditions, the immobilized Hb showed fast and excellent electrocatalytic activity to H2O2 with a small Michaelis-Menten constant of 16 mu mol L-1, a linear range from 2.0 x 10(-6) to 1.1 x 10(-3) mol L-1, and a detection limit of 6.6 x 10(-7) mol L-1. The biosensor also exhibited other advantages, good reproducibility, and long-term stability, and PdNPs/GR-CS nanocomposites film would be a promising material in the preparation of third generation biosensor.