An enzymatic biosensor has been developed for detection of hydrogen peroxide with immobilized Horseradish peroxidase (HRP). HRP was immobilized by using glutaraldehyde (GA) that cross linked with modified polyaniline (PANI) as mediator to improve electron transfer. Modified carbon paste electrodes (MCPE) PANI was more effective during electron transfer compared to carbon paste electrodes (CPE). Cyclic voltammetry method (VC) was used to determine the electrochemical properties of the modified electrode substrate to produce redox reactions. The effect of pH and temperature were analyzed by cyclic voltammetry. The optimum performance of HRP/GA/PANI was at pH 7 and 50°C. Kinetics parameters HRP enzyme were determined in optimum condition. The Michaelis-Menten constant (Km) value and current maximum (I max ) have been obtained as 1.71 mM and 0.29 mA.