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MWCNTs/GR modified electrode which contains multi-walled carbon nanotubes (MWCNTs) and glutathione reductase (GR) has been synthesized on gold electrode. The presence of MWCNTs enhances the surface coverage concentration (Γ) and stability. The measured contact angle of treatment MWCNTs indicates that the sample had more hydrophilic on modified electrode surface. The modified electrode also exhibits a promising enhanced electrocatalytic activity towards the reduction of glutathione. The electrochemical activity of MWCNTs/GR modified gold electrode has been examined using electrochemical impedance spectroscopy (EIS). The cyclic voltammetry (CVs) has been used for the measurement of electroanalytical properties of analytes by means of modified electrodes. The sensitivity values of MWCNTs/GR modified gold electrode are higher than the values which are obtained for only GR modified electrode. We have studied the surface morphology of the ITO modified electrode using scanning electron microscopy (SEM) and atomic force microscopy (AFM), which revealed that GR is coated on MWCNTs. Finally, amperometric response has been used for detection of analytes at MWCNTs/GR film modified gold electrode