In this paper, an amperometric electrochemical biosensor for the detection of hydrogen peroxide (H2O2), based on gold nanoparticles (GNPs)/thionine (Thi)/GNPs/multi-walled carbon nanotubes (MWCNTs)-chitosans (Chits) composite film was developed. MWCNTs-Chits homogeneous composite was first dispersed in acetic acid solution and then the GNPs were in situ synthesized at the composite. The mixture was dripped on the glassy carbon electrode (GCE) and then the Thi was deposited by electropolymerization by Au-S or Au-N covalent bond effect and electrostatic adsorption effect as an electron transfer mediator. Finally, the mixture of GNPs and horseradish peroxidase (HRP) was assembled onto the modified electrode by covalent bond. The electrochemical behavior of the modified electrode was investigated by scanning electron microscope, cyclic voltammetry and chronoamperometry. This study introduces the in situ-synthesized GNPs on the other surface of the modified materials in H2O2 detection. The linear response range of the biosensor to H2O2 concentration was from 5 x 10(-7) mol L-1 to 1.5 x 10(-3) mol L-1 with a detection limit of 3.75 x 10(-8) mol L-1 (based on S/N = 3). (C) 2011 Elsevier B.V. All rights reserved.