Electropolymerized film of Meldola's blue (MB) was prepared and demonstrated as electron shuttle between the immobilized horse peroxidase (HRP) and glassy carbon electrode (GCE) for sensing hydrogen peroxide (H 2 O 2 ) produced by enzyme catalytical reactions. Electrochemical polymerization of Meldola's blue was carried out by cyclic voltammetry (CV) in a phosphate buffer solution (pH 7.00) in a potential window from -0.60 to +1.30 V. The pH of the electropolymerization solution was found to be closely related to the resulted polymeric MB and the best polymeric film was obtained in a pH 7.00 phosphate buffer. The polymeric MB was demonstrated to shuttle the electron transfer between the immobilized HRP and GCE and utilized as a mediator for HRP immobilized biosensor for biocatalytical reduction of H 2 O 2 at a potential of -0.30 V (versus AgCl/Ag). The H 2 O 2 sensing system was applied to construct glucose and choline on-line sensors by wiring H 2 O 2 produced by enzyme oxidase catalytical reaction. The possibility of these sensors as on-line detectors for on-line and continuous measurement was explored off-line. The operating potential, interference, and lifetime of these sensors were also examined.