Gold nanoelectrode ensembles were produced by electrodeposition using multiwalled carbon nanotubes (MWNTs) as template. A new third generation amperometric biosensor for hydrogen peroxide was developed based on adsorption of horseradish peroxidase (HRP) at the glassy carbon (GC) electrode modified with Au nanoelectrode ensembles/multiwalled carbon nanotubes/chitosan film. The resulting HRP biosensor offered an excellent detection for hydrogen peroxide at −0.11 V with a linear response range of 2.08 × 10 −7 to 7.6 × 10 −3 M with a correlation coefficient of 0.998, and response time <5 s. The detection limit was 1.02 × 10 −7 M at 3 σ. The biosensor displays rapid response, expanded linear response range, and excellent repeatability. The simple and fast fabrication of the sensor makes it superior to other techniques.