Composite material based on room temperature ionic liquid (RTIL) N-butylpyridinium hexafluorophosphate (BPPF(6)), sodium alginate (SA), and graphite was used to construct a novel horseradish peroxidase (HRP) biosensor for the determination of H(2)O(2). The morphology of the as-prepared biosensor was characterized by scanning electron microscopy (SEM). Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to characterize the process of the performance of the biosensor. Parameters affecting the performance of the biosensor, including the concentrations of o-aminophenol (OAP) and HRP and the pH value of substrate solution, were optimized. Under the optimized experimental conditions, H(2)O(2) could be detected in a linear calibration range of 1.0 to 6.0 microM with a correlation coefficient of 0.9847 (n=7) and a detection limit of 0.5 microM at a signal/noise ratio of 3. The prepared biosensor not only had economic and disposable property but also showed good detection precision, bioactivity, storage stability, and reproducibility.