Mathanobatins (Mb, Mbtins) were immobilized successfully on nanometer-sized gold colloid particles associated with β-mercaptoethylamine. The structures of Mb functionalized gold nanoparticles were characterized and confirmed by UV-vis spectroscopy (UV-vis), FTIR spectra and electrochemical analyses. Direct electron transfer between Mb or copper-loading Mbtins and the modified electrode was investigated without the aid of any electron mediator. The copper-loading Mbtins act as a better electrocatalyst for the reduction of H₂O₂ than Mb. The copper-loading Mb, with which gold nanoparticles were functionalized, as a model enzyme, was immobilized on gold electrode to construct a novel H₂O₂ biosensor. In pH 6.4 phosphate buffer solution, the reduction and oxidation peak potentials of Mb functionalized gold nanoparticles modified Au electrode (copper-loading Mbtins) were 0.115 and 0.222 V. On the surface, capacitance per unite area (C_d) of Mb functionalized gold nanoparticles modified electrode were 38 μF cm⁻². The immobilized Mb displayed the features of a peroxidase and gave an excellent electrocatalytic response to the reduction of H₂O₂. The detection limit of Mb functionalized gold nanoparticles (copper-loading) were 09 × 10⁻⁵ mA/M (S/N = 3). The Michaelis–Menten constant (Km) was 0.787 mM. Good stability and sensitivity were assessed for the biosensor.