A new electrochemical paper-based analytical device (ePAD) based on cellulose nanofibers (CNs) was developed for the determination of glucose concentration from whole blood samples. The ePAD was fabricated using cellulose acetate (CA) nanofibers and reduced graphene oxide (rGO) nanomaterials for the first time. A smooth and continuous CNs layer was achieved by treating the CA layer with trimethyl chitosan (TMC). Glucose oxidase was immobilized onto the regenerated CNs layer in the sensing area of the rGO-modified screen-printed three electrodes (SPEs). The ePAD glucose assay showed a high sensitivity of 9.9 × 10 -4 KΩ -1 •mM-1 to glucose in the range 3.3 -27.7 mM, with a detection limit of 0.1 mM, high reproducibility, and outstanding selectivity and stability. The device demonstrated excellent analytical activity toward glucose determination, and its low cost and user-friendly design make it a promising tool the detection of other biomolecules.