FAD-dependent glucose dehydrogenase (FAD-GDH) of Burkholderia cepacia was successfully expressed in Escherichia coli and subsequently purified in order to use it as an anode catalyst for enzyme fuel cells. The purified enzyme has a low Km value (high affinity) towards glucose, which is 463.8 μM, up to 2-fold exponential range lower compared to glucose oxidase. The heterogeneous electron transfer coefficient (Ks) of FAD-GDH–menadione on a glassy carbon electrode was 10.73 s−1, which is 3-fold higher than that of GOX–menadione, 3.68 s−1. FAD-GDH was able to maintain its native glucose affinity during immobilization in the carbon nanotube and operation of enzyme fuel cells. FAD-GDH–menadione showed 3-fold higher power density, 799.4 ± 51.44 μW cm−2, than the GOX–menadione system, 308.03 ± 17.93 μW cm−2, under low glucose concentration, 5 mM, which is the concentration in normal physiological fluid.