This study demonstrates the capability of graphene as a spacer to form eletrochemically functionalized multilayered nanostructures onto electrodes in a controllable manner through layer-by-layer (LBL) chemistry. Methylene green (MG) and positively Charged methylimidazolium-functionalized multiwalled:carbon nanotubes (MWNTs) were used as examples of electroactive species and electrochemically useful components for the assembly, respectively. By using graphene as the spacer, the multilayered nanostructure of graphene/MG and graphene/MWNT could be readily formed onto electrodes with the LBL method on the basis of the electrostatic and/or pi-pi interaction(s) between graphene and the electrochemically useful components. Scanning electron microscopy (SEM) ultraviolet-visible spectroscopy (UV-vis), and cyclic voltammetry (CV) were used to characterize the assembly processes and the results revealed that nanostructure assemble was uniform and effective with graphene as the spacer Electrochemical studies demonstrate that the assembled nanostructures posses excellent electrochemical properties and electrocatalytic activity toward the oxidation of NADH and could thus be used as electronic transducers for bioelectronic devices. This potential was further demonstrate by using an alcohol dehydrogenase-based electrochemical biosensor and glucose dehydrogenase-based glucose/O(2) bioduel cell as typical examples. This study offers a simple route to the controllable formation of graphene-based electrochemically functionalized nanostructures that can be used for the development of molecular bioelectronic devices such as biosensors and biofuel cells.