In this work, a new glucose amperometric biosensor was developed by directly electrospinning poly(vinyl alcohol)/chitostan nanofibers on the surface of the platinum electrode, in which glucose oxidase (GOD) was effectively immobilized in nanofibers by encapsulation. After been cross-linked in glutaraldehyde vapor and modified with a thin nafion film, the nanofibers (PVA/chitosan/GOD)/nafion electrode was used for glucose amperometric measurements. The electrospun nanofibrous enzyme membrane served as a better sensing element than the casing one due to the unique properties of nanofibers such as the special three-dimensional network structure, large pores, high porosity, and large surface to volume ratios. The as-prepared biosensor showed a wide linear calibration range, low detection limit, and low apparent Michaelis-Menten constant in the glucose determination. The stability, reproducibility and anti-interference capability of biosensor were also presented. Furthermore, the new biosensor was successfully applied to detect glucose in human serum samples.