<p>Immobilization of electro-functional molecules onto the electrode surface often plays an important role in the fabrication of electrochemical devices such as photo-voltaic cells, fuel cells, semiconductors, and sensors. Although some molecular-immobilization techniques have been developed, few studies have explored relationships between the immobilization techniques and the properties of the resulting composite electrodes such as the surface density of the molecules, charge transfer rate between the electrode and molecules. In this study, electroactive Fc molecules were immobilized on an ITO electrode surface by two different immobilization methods, i.e., (1) azido functionalization of the ITO surface with 3-azidopropyltriethoxysilane (N₃-PTES) followed by copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) with ethynylferrocene (ITO|N₃-PTES|Fc) and (2) azido functionalization of the ITO surface with electro-deposited 2′-azidomethyl-3,4-ethylenedioxythiophene (N₃-EDOT) polymer film followed by CuAAC with ethynylferrocene (ITO|N₃-EDOT|Fc). A characteristic difference in the electrochemical devices fabricated by the two methods was the rate of electron transfer at the immobilized Fc interface. The apparent charge transfer constant on the ITO|N₃-EDOT|Fc was estimated to be 2.8&#xa0;s⁻¹, while that on the ITO|N₃-PTES|Fc was much faster at 97&#xa0;s⁻¹. This suggests that molecular immobilization methods such as silane coupling agents, which tend to give monomolecular layer, offer efficient electron transfer compared to electropolymerization, which tends to give random and thick molecular layer. These results suggest that different immobilization methods of ITO substrates can significantly affect the charge transfer efficiency of immobilized Fc, demonstrating that they provide important guidelines for the fabrication of more efficient electrochemical devices.</p> Graphical abstract <p></p>