A facile strategy to construct an amperometric biosensor was described for the determination of hydrogen peroxide (H2O2). This biosensor relied on an electrospinning gold nanoparticle-chitosan-poly(vinyl alcohol) composite nanofibers modified ITO electrode, followed by immobilization of hemoglobin (Hb) on the surface. The introduction of nanofibers and gold nanoparticles in the modification of electrode surface not only enhanced the surface area of the modified electrode for enzyme immobilization but also facilitated the electron transfer rate. Under optimum conditions, the sensor was characterized in terms of its morphology by scanning electron microscopy and its electroactivity by cyclic voltammetry and chronoamperometry. Scanning electron microscopy revealed that the obtained nanofibers were uniform. The chronoamperometric behavior of the modified electrode indicated that the immobilized Hb retained electrochemical activity inside the electrospinning fibrous membranes. The electrode responded linearly to H2O2 in a wider concentration range of 5.6x107M to 5.2x102M with a low detection limit (S/N=3) of 1.98x107M and a short response time of approximate to 4s, suggesting a much better performance than that of other sensors. Moreover, the biosensor achieved bulk production and exhibited superior properties for the sensitive determination of H2O2, studied namely, long-term stability, good reproducibility, and high selectivity.