Production of multifunctional biomaterials in nanoscale is one of the most popular topics in nanotechnology related to biological applications because of its biocompatibility and biodegradability. Herein, cellulose acetate (CA) was blended with chitosan (CS) for the formation of non-soluble electrospun nanofibers in aqueous medium. Amine-functionalized nanofiber surfaces were obtained using carbohydrates, which is an advantage for biomolecule immobilization by covalent bonds. Different solvent systems and CA-CS feeding ratio were examined to obtain ultrafine non-beaded nanofibers. After showing the surface characteristics of CA-CS nanofibers, glucose oxidase (GOx) was immobilized on CA-CS coated electrode by covalent conjugation. Cyclic voltammetry, differential pulse voltammetry, and electrochemical impedance spectroscopy measurements were carried out to prove the surface modification. Finally, CA-CS/GOx platform was investigated to detect glucose in samples and some potential interfering compounds were tested to find out the performance of CA-CS/GOx. CA-CS electrospun nanofibers were well-characterized and applied successfully to fabricate repeatable electrochemical enzymatic biosensor for the first time in this paper.