Hydrophobins are a family of natural self-assembling proteins with high biocompability, which are apt to form strong and ordered assembly onto many kinds of surfaces. These physical-chemical and biological properties make hydrophobins suitable for surface modification and biomolecule immobilization purposes. A class II hydrophobin HFBI was used as enzyme immobilization matrix on platinum electrode to construct amperometric glucose biosensor. Permeability of HFBI self-assembling film was optimized by selecting the proper HFBI concentration for electrode modification, in order to allow H(2)O(2) permeating while prevent interfering compounds accessing. HFBI self-assembly and glucose oxidase (GOx) immobilization was monitored by quartz crystal microbalance (QCM), and characterization of the modified electrode surface was obtained by scanning electron microscope (SEM). The resulting glucose biosensors showed rapid response time within 6s, limits of detection of 0.09 mM glucose (signal-to-noise ratio=3), wide linear range from 0.5 to 20mM, high sensitivity of 4.214 x 10(-3)AM(-1)cm(-2), also well selectivity, reproducibility and lifetime. The all-protein modified biosensor exhibited especially high efficiency of enzyme utilization, producing at most 712 microA responsive current for per unit activity of GOx. This work provided a promising new immobilization matrix with high biocompatibility and adequate electroactivity for further research in biosensing and other surface functionalizing.