A new type of hydrogen peroxide amperometric biosensor was fabricated based on electrochemically deposited sub-micrometer Au particles (sm-Au) on a glassy carbon electrode (GCE). Electrochemical deposition condition was optimized for obtaining uniformly distributed sub-micrometer sized Au array on the electrode surface. The hy-drogen peroxide sensor was fabricated by adsorbing phenothiazine methylene blue (MB) molecules on the surface of sm-Au and covering a cross-linked horseradish peroxidase (HRP) layer, labeled as HRP/MB/sm-Au/GCE. The characteristics of this biosensor were evaluated with respect to applied potential and pH. The amperometric re-sponse of the sensor was linear to the H2O2 concentration over a wide range of 9.9×10-61.11×10-2 mol/L. A detection limit (s/n=3) of 3.0×10-6 mol/L H2O2 was estimated for a sampled chronoamperometric detection at 1.5 min after potential step of 200 to -400 mV vs. SCE. The immobilized MB molecules shuttled electrons at a=0.77 and an apparent electron transfer rate constant of 0’sk=0.053 s-1. Interference of ascorbic acid, dopamine and uric acid was investigated. This sensor has very good stability and reproducibility for long-term use. A new type of hydrogen peroxide amperometric biosensor was fabricated based on electrochemically deposited sub-micrometer Au particles (sm-Au) on a glassy carbon electrode (GCE). Electrochemical deposition condition was optimized for uniformly distributed distributed sub-micrometer sized Au array on the The hy-drogen peroxide sensor was fabricated by adsorbing phenothiazine methylene blue (MB) molecules on the surface of sm-Au and covering a cross-linked horseradish peroxidase (HRP) layer, labeled as HRP / MB / GCE. The characteristics of this biosensor were evaluated with respect to applied potential and pH. The amperometric re-sponse of the sensor was linear to the H2O2 concentration over a wide range of 9.9 × 10-61.11 × 10-2 mol / L. A detection limit (s / n = 3) of 3.0 × 10 -6 mol / L H 2 O 2 was estimated for a sampled chronoamperometric detection at 1.5 min after potential step of 200 to -400 mV vs. SCE. The immobilized MB molecules shuttled electrons at a = 0.77 and an apparen t electron transfer rate constant of 0’sk = 0.053 s-1. Interference of ascorbic acid, dopamine and uric acid was investigated. This sensor has very good stability and reproducibility for long-term use.