A novel glucose oxidase (GOD) biosensor was fabricated with a protic ionic liquid (PIL) N-ethylimidazolium trifluoromethanesulfonate ([EIm][TfO]) as the modifier of a carbon electrode. Due to the excellent conductivity and the conformational changes of the microenvironment around the GOD, the electrochemical and biocatalytic properties of GOD immobilized on the PIL-based electrode were dramatically enhanced. A couple of well-defined redox peaks could be observed, with a formal potential of −0.476 V. The GOD biosensor presented good catalytic activity to the oxidation of glucose in oxygen-saturated phosphate buffer solutions. The cathodic peak currents of GOD decreased along with glucose concentrations. A linear response in the range 0.005–2.8 mM was obtained with a detection limit of 2.5 μM. The sensitivity and the apparent Michaelis–Menten constant (K m) were estimated to be 14.96 μA mM−1 and 1.53 μM, respectively. In addition, the biosensor remained stable over 30 days, indicating its good chemical and mechanical stability. The glucose content of several serum samples was determined by using the newly developed biosensor, and the results were in good agreement with those obtained by hospital measurements. All results suggested that PILs were a good media for supporting biocatalytic processes on the bioelectrode.