Nanofibrous membranes have been produced by electrospinning to develop first generation glucose biosensors. The direct immobilization of glucose oxidase onto the polyamide nanofibrous surfaces by drop coating revealed a simple and efficient method for the development of sensitive, stable, and reproducible electrochemical biosensors. The biosensor showed a linear response over the range 1–9×10−3 glucose (R2=0.9997) with a sensitivity of 1.11 μA/mM and a limit of detection of 2.5×10−6 M (S/N=3). The uncertainty of repeatability was 2% (RSD%, n=30). After one month of storage, the signal decreased of 35%. The recovery of glucose, evaluated in real samples of honey, was 98% (RSD%=1%, n=3).