A novel fabrication approach for the amperometric biosensor composed of carbon nanotubes (CNT), a plasma-polymerized film (PPF), hexamineruthenium(III)chloride (RU), and enzyme glucose oxidase (GOD) is reported. The configuration of the electrochemical electrode is multilayer films which contain sputtered gold, lower acetonitrile PPF, CNT, RU, GOD, and upper acetonitrile PPF, sequentially. First, PPF deposited on Au acts as a permselective membrane and as a scaffold for CNT layer formation. Second, PPF directly deposited on GOD acts as a matrix for enzyme immobilization. To facilitate the electrochemical communication between the CNT layer and GOD, CNT was treated with nitrogen plasma. The electron transfer mediator RU play a role as the mediator, in which the electron caused by enzymatic reaction transports to the electrode. The synergy between the electron transfer mediator and CNT provides benefits in terms of lowering the operational potential and enhancing the sensitivity (current). The optimized glucose biosensor revealed a sensitivity of 3.4µA mM^-1 cm^-2 at +0.4V vs. Ag/AgCl, linear dynamic range of 2.5-19mM, and a response time of 6s.