Horseradish peroxidase (HRP)-containing multilayer films have been fabricated by alternate deposition with bipolar pyridine salt (PyC6BPC6Py) on the surface of gold electrode derivatized by 3-mercapto-1-proponesulfonate monolayer. The driving force of forming the multilayer structures is the electrostatic interaction between the negatively charged HRP and the positively charged PyC6BPC6Py. The assembly process was monitored by ultraviolet-visible spectroscopy on quartz slides. The electrochemical behavior of the enzyme electrode showed that the multilayer film was stable and electroactive. The diffusing mediator, methylene green in solution as an electron shuttle, was able to Transfer electrons successfully between the active center of the immobilized HRP and the electrode surface. Its potential application as amperometric sensor for hydrogen peroxide is anticipated.