A novel two-step method is presented for the electropolymerization of thionine in neutral phosphate buffer solutions containing 1 x 10(-4) M thionine on glassy carbon electrode surfaces. The formation of the PTH film is dependent on the preanodization voltage and occurs only on a GCE surface, The preanodization operation is critical. During this period, a large amount of positive charge can be accumulated on the GCE surface and is used to create the thionine cation radical for the electropolymerization of thionine. The poly(thionine) (PTH)-modified electrode obtained in this manner has a golden, fairly even film, which is firm and permanent. Strong treatment in an ultrasonic bath for 20 min does not affect the voltammetric redox peaks of PTH. The E-0＇ decreases by 55 mV/pH between pH 6 and 8 for the immobilized PTH, which is close to the expected Nernstian value of 59 mV for a two-electron, two-proton process. The PTH-modified electrodes show linear dependence of peak current with scan rate within the range of 20 to 100 mV s(-1). A H2O2 biosensor was fabricated by using Nafion membranes with enzyme. The catalytic reaction towards H2O2 reduction occurring on the H2O2 sensor in the presence of 1 mM H2O2 demonstrated that the film of PTH can effectively shuttle electrons between the redox center of HRP and the base electrode.