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In order to enhance the performance of the Direct Methanol Fuel Cell (DMFC), the product of CO2 bubble has to be efficiently removed from the anode channel during the electrochemical reaction. In this study, the materials of Polymethyl Methacrylate (PMMA) with hydrophilic property and polydimethylsiloxane (PDMS) with hydrophobic property are used to form the anode cannel. The channel of the DMFC is fabricated through a microelectromechanical system (MEMS) manufacture process. Two DMFC are stacked together with a common cathode channel for reducing the volume of the cells, and form a twin stack DMFC. The performance of the twin stack DMFC is investigated under the influence of operation conditions, including operation temperature and flow rate. The twin stack DMFC, whose anode channel is made of PDMS possess the best performance among the twin stack DMFC under any of the specific operating conditions tested. However, the increase in the operation temperature and fuel flow rate resulted in decreased the hydro-resistance reduction effect of PDMS, and the performance enhancement of the PDMS DMFC is not obvious in the operating condition of high temperature and high flow rate.