A new biosensor based on catalase enzyme immobilized on electrochemically constructed polyaniline (PANI) film modified with glutaraldehyde has been developed for the determination of hydrogen peroxide (H2O2) in milk samples. Assembly processes of polyaniline and immobilization of the enzyme were monitored with the help of electrochemical impedance spectroscopy. Amperometric measurements have been performed at cathodic peak (-0.3 V vs. Ag/AgCI) which was attributed to reduction of PANI. Hydrogen peroxide was determined by using amperometric method at -0.3 V. The biosensor responses were correlated linearly with the hydrogen peroxide concentrations between 5.0 x 10(-6) and 1.0 x 10(-4) M by amperometric method. Detection limit of the biosensor is 2.18 x 10(-6) M for H2O2. In the optimization studies of the biosensor, some parameters such as optimum pH, temperature, concentration of aniline, amount of enzyme, and number of scans during electropolymerization were investigated.