A novel technique of applying electrochemical impedance sensor for the detection of Vitamin B12 is described in this study.The impedance biosensor was fabricated by immobilizing Vitamin B12 on a gold electrode assembled with 11- mercaptopropionic acid monolayer.The processes of functionalized Au electrode were characterized by electrochemical impedance spectroscopy and cyclic voltammetry.Faradic impedance spectra measurements for the determination of Vitamin B12 concentration were performed based on the charge transfer mechanisms of the redox probe of [Fe (CN) 6]3-/4-.The diameter of Nyquist diagram that is equal to the charge transfer resistance (Rct) increases with the increase of the concentration of Vitamin B12.A linear relationship between the Rct values and logarithmic magnitude of Vitamin B12 concentration was found in the range of 10-10 and 10-5 M.