We developed glucose biosensing electrodes using single-walled carbon nanotube (SWCNT) films on flexible, transparent poly(ethylene terephthalate). The homogeneous SWCNT films were fabricated by a vacuum filtration method, and the averaged resistivity and transparency of the fabricated flexible SWCNT films were 400 Ω sq −1 and 80%, respectively. The glucose sensing electrodes were constructed by encapsulating glucose oxidase (GOx) by Nafion binder into the SWCNT film, and the variation in current response as a function of enzyme loading amount, Nafion thickness were investigated. 30 mg mL −1 GOx and 2% Nafion was optimal for the detection of glucose. When ferrocene monocarboxylic acid (FMCA) was introduced as diffusional electron mediator, the current responses toward glucose of the Nafion/GOx/SWCNT electrodes in glucose solution containing FMCA were dramatically improved, and the developed sensor was independent of oxygen. In the application of GOx immobilized SWCNT films for glucose detection, a linear electrical response was observed for concentrations ranging from 0.25 to 3.0 mM, and the detection limit and the sensitivity were assessed to be 97 μM and 9.32 μA mM −1 cm −2, respectively. Moreover, according to the Lineweaver–Burk plot, the apparent Michaelis–Menten constant was calculated to be 23.8 mM, and the current responses did not interfere with coexisting electroactive species, indicating that Nafion is an effective permselective polymer barrier.