The combination of nanomaterials and conducting polymers attracted remarkable attention for development of new immobilization matrices for enzymes. Hereby, an efficient surface design was investigated by modifying the graphite rod electrode surfaces with one-step electrospun nylon 6,6 nanofibers or 4% (w/w) multiwalled carbon nanotubes (MWCNTs) incorporating nylon 6,6 nanofibers (nylon 6,6/4MWCNT). High-resolution transmission electron microscopy study confirmed the successful incorporation of the MWCNTs into the nanofiber matrix for nylon 6,6/4MWCNT sample. Then, these nanofibrous surfaces were coated with a conducting polymer, (poly-4-(4,7-di(thiophen-2-yl)-1H-benzo[d]imidazol-2-yl)benzaldehyde) (PBIBA) to obtain a high electroactive surface area as new functional immobilization matrices. Due to the free aldehyde groups of the polymeric structures, a model enzyme, glucose oxidase was efficiently immobilized to the modified surfaces via covalent binding. Scanning electron microscope images confirmed that the nanofibrous structures were protected after the electrodeposition step of PBIBA and a high amount of protein attachment was successfully achieved by the help of high surface to volume ratio of electroactive nanofiber matrices. The biosensors were characterized in terms of their operational and storage stabilities and kinetic parameters (K-m(app) and I-max). The resulting novel glucose biosensors revealed good stability and promising I-max (10.03 and 16.67 mu A for nylon 6,6/PBIBA and nylon 6,6/4MWCNT/PBIBA modified biosensors, respectively) and long shelf life (32 and 44 days for nylon 6,6/PBIBA and nylon 6,6/4MWCNT/PBIBA modified biosensors, respectively). Finally, the biosensor was tested on beverages for glucose detection.