A new electrospinning method has been developed to create a copper-nanoflower decorated gold nanoparticle-graphene oxide (Cu-NF@AuNPs-GO) nanofiber for use as an electrochemical biosensor to detect glucose. Graphene oxide mixed with poly(vinyl alcohol) was used as a fiber precursor to improve electrochemical properties, with the solution being coated onto gold chips to create uniform GO nanofibers via electrospinning. Gold nanoparticles were coated onto the GO nanofibers, which were then integrated with an organic-inorganic hybrid nanoflower containing copper nanoflower-glucose oxidase and horseradish peroxidase. The resulting Cu-NF@AuNPs-GO nanofibers showed excellent electrochemical catalytic properties and selectivity for converting glucose to gluconic acid in the presence of GOx-HRP-Cu nanoflower, with a linear range of 0.001-0.1 mM and a detection limit of 0.018 μM. The modified Au chip exhibited superior catalytic properties due to the coating of unique organic-inorganic nanostructured materials on its surface.