Micro- and nanofiber (NF) hydrogels fabricated by electrospinning to typically exhibit outstanding high porosity and specific surface area under hydrated conditions. However, the high crystallinity of NFs limits the achievement of transparency via electrospinning. Transparent poly(vinyl alcohol)/β-cyclodextrin polymer NF hydrogels contacted with reverse iontophoresis electrodes were prepared for the development of a non-invasive continuous monitoring biosensor platform of interstitial fluid glucose levels reaching ~ 1 mM. We designed the PVA/BTCA/β-CD/GOx/AuNPs NF hydrogels, which exhibit flexibility, biocompatibility, excellent absorptivity (DI water: 21.9 ± 1.9, PBS: 41.91 ± 3.4), good mechanical properties (dried: 12.1 MPa, wetted: 5.33 MPa), and high enzyme activity of 76.3%. Owing to the unique features of PVA/β-CD/GOx containing AuNPs NF hydrogels, such as high permeability to bio-substrates and rapid electron transfer, our biosensors demonstrate excellent sensing performance with a wide linear range, high sensitivity(47.2 μA mM−1), low sensing limit (0.01 mM), and rapid response time (< 15 s). The results indicate that the PVA/BTCA/β-CD/GOx/AuNPs NF hydrogel patch sensor can measure the glucose concentration in human serum and holds massive potential for future clinical applications.