In this paper, we report a novel and efficient bioactive surface nanostructuration method using enzyme-based electrospun nanofibers decorated with gold nanoparticles (Au NPs) for electrochemical biosensors application. Poly(vinyl alcohol)/poly(ethyleneimine) nanofibers (PVA/PEI NFs) were first produced at the surface of gold electrodes in a one-step and rapid electrospinning process. Different parameters such as electrospun solution feed rate, applied voltage and distance between tip and collector were tailored to produce NFs with minimal beading. Scanning electron microscopy (SEM) was used to characterize their morphology. It was shown that adhesion of the fibers mat onto the electrodes could be improved by modifying the surface with a self-assembled monolayer of 4-aminothiophenol (4-ATP), bearing thiol groups for covalent binding to the gold surface and amine groups to react with the amine groups of PEI in a subsequent cross-linking step using glutaraldehyde vapors. This step not only helped anchoring the NFs onto the electrode but also improved their water stability. The electrochemical properties of electrospun PVA/PEI nanofibrous mats were characterized by cyclic voltammetry and electrochemical impedance spectroscopy. The NFs conductive properties could be significantly improved by decorating them with AuNPs. The highest density of particles was observed using Au colloidal solutions of pH 5, which could be attributed to hydrogen bondings and ionic interactions between the amine groups of PEI and the carboxylic groups of citrate-stabilized Au NPs. Glucose oxidase (GOx), used as model enzyme, was further incorporated into the PVA/PEI mixture, before electrospinning, resulting in the fabrication of 4-ATP/PVA/PEI/GOx NFs. Enzyme activity was preserved and the obtained biosensor enabled successful detection of glucose by electrochemical impedance spectroscopy. This novel glucose biosensor exhibited a good stability, the response was linear in the 10–200 μM range and a very low limit of detection (0.9 μM) could be achieved.