Objective To construct glucose determination biosensor with immobilized glucose oxidase and evaluate the characteristic of biosensor.Methods Prepared immobilized glucose oxidase with good performance by optimizing the immobilized carriers and the immobilization conditions of glutaral cross-linking.The characteristic of immobilized enzyme was studied,and used to construct glucose biosensor.Results The optimal catalytic temperature and pH of immobilization glucose oxidase were 40 ℃ and 7.0,respectively.The response time of glucose biosensor was less than 20 s,the linear range of glucose concentration was 0~1.5 g·L-1.and had high anti-interference and stability.Conclusion The integrated glucose determination biosensor had good performance and can be used for the determination of glucose in biological samples.