© 2021 Elsevier B.V.In this study, designing of a stable electrospun blended chitosan (CS)-poly(vinyl alcohol) (PVA) nanofibers for colorimetric glucose biosensing in an aqueous medium was investigated. CS and PVA solutions were blended to acquire an optimum content (CS/PVA:1/4) and electrospunned to obtain uniform and bead-free CS/PVA nanofiber structures following the optimization of the electrospinning parameters (33 kV, 20 cm, and 1.2 ml.h−1). Crosslinking process applied subsequently provided mechanically and chemically stable nanofibers with an average diameter of 378 nm. The morphological homogeneity, high fluid absorption ability (>%50), thermal (<230 °C) and morphological stability, surface hydrophilicity and degrability properties of cross-linked CS/PVA nanofiber demonstrated their great potential to be developed as an eye-readable strip for biosensing applications. The glucose oxidase (GOx) and horseradish peroxidase (HRP) was immobilized by physical adsorption on the cross-linked CS/PVA nanofiber. The glucose assay analysis by ultraviolet-visible (UV–Vis) spectrophotometry using the same enzymatic system of the proposed glucose strips in form of absorbance versus concentration plot was found to be linear over a glucose concentration range of 2.7 to 13.8 mM. The prepared naked eye colorimetric glucose detection strips, with lower detection limit of 2.7 mM, demonstrated dramatic color change from white (0 mM) to brownish-orange (13.8 mM). The developed cross-linked CS/PVA nanofiber strips, prepared by electrospinnig procedure, could be easily adapted to a color map, as an alternative material for glucose sensing. Design of a practical, low-cost, and environmental-friendly bio-based CS/PVA testing strips for eye readable detection were presented and suggested as an applicable medium for a wide range of glucose concentrations.