A novel nanocomposite designed by the assembly of the positively charged poly(diallyldimethylammonium chloride) protected gold nanoparticles (PDDA-GNPs), and the negatively charged multi-walled carbon nanotubes (MWCNTs) on ITO electrode via electrostatic interaction, was used as a supporting matrix for immobilizing hemoglobin (Hb) to develop a high-performance hydrogen peroxide (H 2 O 2 ) biosensor. The cyclic voltammetrys of immobilized Hb showed a pair of well-defined and quasi-reversible redox peaks with the formal potential of -0.205V (vs. SCE) and the peak-to-peak potential separation of 44 mV at a scan rate of 100 mV·s -1 in 0.1 mol×L -1 pH 7.0 PBS. Under the optimized experimental conditions, a linearity range for determination of H 2 O 2 was from 2.0 × 10 -6 to 5.2 × 10 -4 mol×L -1 with a correlation coefficient of 0.9994 (n = 37) and a detection limit of 8.4 × 10 -7 mol×L -1 . The biosensor displayed excellent electrochemical and electrocatalytic response to the reduction of H 2 O 2 , high sensitivity, long-term stability, good bioactivity and selectivity.