Abstract: Cytochrome C and mercaptopropionic acid modified Au electrode (Cyt. C/MPA/Au) and hexa-(ferrocene)-hexyl mercaptan modified Au electrode (FcHT/Au) were prepared, and the electrochemical property of two modified electrodes was studied by cyclic voltammetry. It was found that in PBS of pH 7.0, the reduction peak current of H₂O₂ at the Cyt. C/MPA/Au increased with the increase of the content of H₂O₂, while the reference peak current at the FcHT/Au remained basically unchanged. Based on this finding, a double-channel ratiometric H₂O₂ biosensor was proposed. Content of H₂O₂ was determined by DPV. Linear relationship between values of peak current ratio of H₂O₂ at Cyt. C/MPA/Au to that at FcHT/Au and concentration was ranged from 20.0-100.0 μmol·L^-1, with detection limit (3S/N) of 1.19 μmol·L^-1. Recovery of the method was in the range of 71.4%-79.2%.