Fenton oxidation method using hydrogen peroxide is an eco-friendly oxidation method used in water treatment and soil restoration. When removing pollutants by this method, it is quite important to properly regulate the concentration of hydrogen peroxide according to the concentration of the contaminants. In this study, electrochemical biosensors using HRP (horseradish peroxidase) enzymes were manufactured and studies were conducted on the activity of enzymes and the detection characteristics of hydrogen peroxide. HRP were electro deposited with chitosan and AuNP on the working electrode surface of the SPCE (Screen Printed Carbon Electrode). Then, the fixation of enzymes was confirmed using the cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The activity of HRP enzymes was also identified from chronoamperometry (CA) and UV spectroscopy. After immersing the biosensor in PBS solution the current generated from electrodes by titrating hydrogen peroxide was measured from CA analysis. The generated current increased linearly for the concentration of hydrogen peroxide, and a calibration curve was derived that could predict the concentration of hydrogen peroxide from the current.