Although the uses of alternative energy sources have been increased greatly in recent years, current major energy demand is still supplied from conventional fossil fuels such as oil, coal and natural gas. Considering the inevitable depletion of the world’s energy supply and the release of green-house in the earth’s atmosphere, it is necessary to look for an alternative source of fuel. Alkaline-anion-exchange membrane direct alcohol fuel cells (AAEM-DAFCs) are among the promising candidates as alternative power sources for both stationary and portable power applications. Due to the facile kinetics at both the cathode and the anode in alkaline media, the use of non-noble and low-cost material catalysts become feasible, which makes the AAEM-DAFCs a potentially low cost technology compared with proton exchange membrane (PEM) based DAFCs working in acidic media. Bioethanol has high energy density and is non-toxicity, easy to transport and can be distributed with the existing infrastructure. Bioethanol is a renewable bio-based liquid fuel that can be produced from several different biomass feedstock and conversion technologies. First generation bioethanol is produced by distillation from crops such as wheat, corn, sugar cane and sugar beet; however, this bioethanol production has limitation due to concerns about food price and land use impacts. Second generation bioethanol is produced from different types of lignocellulosic materials as substrate. Currently, only negligible amounts of second generation bioethanol are produced in several demo plants around the world that work industrially, but are not yet commercially feasible. In this work, the bioethanol was produced by diversity feed stocks, like food waste and cellulosic, away from expensive corn. The produced bioethanol may contain some impurities, such as acetic acid, lactic acid, phosphorus, maltotetraose (Dp4), glucose, maltose, fructose, glycerol, isomaltotriose (Dp3), sulfur and so on. Pd/C was a promising catalyst for the ethanol oxidation reaction (EOR) in alkaline media. Our earlier work indicated that the PdRu/C catalyst exhibited much higher initiate activity toward EOR than the Pd/C catalyst in fuel cell operation conditions. Therefore, we further study the tolerance of impurities in bioethanol on Pd/C and PdRu/C catalysts toward EOR in alkaline media and demonstrate their performance and stability in AAEM-DAFCs using bioethanol as the fuel. Acknowledgements We acknowledge financial supports from the National Science Foundation Partnerships for Innovation: Building Innovation Capacity (Contract 1317731).