A novel platform based on reduced graphene oxide and SiO₂/CMC hybrid membranes(GO-SiO₂-CMC) was successfully constructed for the fabrication of biosensor. The morphologies and electrochemistry of the nanocomposite film were investigated by using scanning electron microscopy and electrochemical techniques including electrochemical impedance spectroscopy and cyclic voltammetry (CV), respectively. The results suggested that the modified electrode showed enhanced sensitivity towards the oxidation of Vitamin B6 (VB6), e.g., high detection sensitivity (31.5μAmM-1cm-2), a low limit of detection (10-7M), a small Ret value (252Ω) and a good stability. This might be attributed to large surface-to-volume ratio and high conductivity of graphene, which could promote direct electron transfer between the surface of electrodes and Vitamin B6 (VB6). These above results offered a simple, sensitive and flexible method for quantitative detection of Vitamin B6 through the technology of electrochemical.