This paper reports on the preparation and advantages of novel amperometric biosensors in the presence of hydrophobic ionic liquid (IL), 1-methyl-3-butylimidazolium bromide ([MBIB]). Carbon paste biosensor has been constructed by entrapping horseradish peroxidase in graphite and IL mixed with paraffin oil as a binder. The resulting IL/graphite material brings new capabilities for electrochemical devices by combining the advantages of ILs composite electrodes. Amounts of H2O2 were amperometrically detected by monitoring current values at reduction potential (-0.15 V) of K3Fe(CN)(6). Decrease in biosensor responses were linearly related to H2O2 concentrations between 10 and 100 mu M with 2 s response time. Limit of detection of the biosensor were calculated to be 3.98 mu M for H2O2. In the optimization studies of the biosensor some parameters such as optimum pH, optimum temperature, enzyme amount, interference effects of some substances on the biosensor response, reproducibility and storage stability were carried out. The promising results are ascribed to the use of an ionic liquid, which forms an excellent charge-transfer bridge and wide electrochemical windows in the bulk of carbon paste electrode.