Graphite oxide was prepared by Hummers method. Graphene oxide was obtained from ultrasonic oscillation of graphite oxide and then reduced by using hydrazine hydrate to get graphene (GR). The results showed that the prepared graphene oxide enlarged interlamellar spaces compared to graphite which was from 0.33 nm to about 0.86 nm. The structure of graphene oxide contained many oxygen groups which resulted in the increase of interlamellar space and hydrophilism. After reducion, some oxygen groups disappeared. Hydrogen peroxide biosensor based on graphene modified horseradish peroxidase electrode was prepared by using graphene as enzyme carrier, chitosan (CS) as binder, thionine (Th) as electrical mediator and glass carbon (GC) as electrode matrix. Graphene and Th had the synergistic effect on HRP-GR-Th- CS/GC electrode. The cyclic voltammetric experiment results demonstrated that the current response of the biosensor was linear to hydrogen peroxide content ranging from 5.0×10-5 to 1.1×10-3 mol/L, with detection limit of 1.50×10-6 mol/L and apparent Michaelis-Menten constant ( ) of 6.55×10-5 mol/L. The biosensor has good detection performance especially for hydrogen peroxide thus can be used for detecting hydrogen peroxide content.