Abstract: A novel prototype of nano-structure glucose/O₂ biofuel cell was constructed with immobilized enzyme on 4-mercapto benzoic acid(4-MBA) modified gold nanoparticles as electrical medium and enzyme carrier. It features with simple fabrication and favorable long-term usability. Glucose oxidase(GO_x) and Laccase(Lac) molecules are tethered steadily to the surface of gold nanoparticles via interaction between modified group of gold nanoparticles and hydrophobic binding-sites in the vicinity of cofactor within enzyme.The as-prepared bioanode and biocathode are fabricated and denoted as GO_x/4-MBA@GNP/Au and Lac/4-MBA@GNP/Au,respectively. Electrochemical results indicate that the direct electron transfer occurs between enzyme active sites and gold nanoparticles for both biocathode and bioanode in the absence of any mediator achieves with fast catalytic activity(turn-over frequency of bioanode and biocathode:1.3 and 0.5 s^-1,onset potential according to the glucose catalytic oxidation and catalytic reduction of oxygen:-0.23 and 0.76 V). The power out-put performance of nano-structure with entrapped enzyme glucose/O₂ biofuel cell was further evaluated after the constitution of cell via connection bioanode and biocathode on the basis of previous results. The results of test show that the open-circuit voltage and the maximum out-put energy density of this biocell amount to 0.56 V and 760.0 μW/cm^-2 in the absence of Nafion ion-exchange membrane and anode protection gas of N₂,respectively. The out-put density of biofuel cell after storage in refrigerator for one week can still retain ~88% of the initial value. Furthermore,this fuel cell shares the similar characteristics of pH dependence and thermal stability to those of free laccase. The key factor contributed to the performance of biofuel cell should be related to the catalytic oxygen reduction process at biocahode. Despite of the apparent influence of concomitant interferent-ascorbic acid on cell performance,this cell still shows superior out-put energy density(132.0 μW/cm^-2,open circuit voltage:0.40 V) recorded in the circumstance of the human serum. This study may afford a new route to design in high performance glucose/O₂ fuel cell and provide experimental basis and valuable enlightenment for the study of relationship between the structure of enzyme based cell and its performance.