Quantification of the blood glucose concentration in the whole blood was not easy to achieve because the detection process was affected by many factors, such as glucose metabolism and biofouling. In this paper, we established an amperometric glucose biosensor applied in whole blood directly, which was based on the direct electron transfer of glucose oxidase (GOx) entrapped onto the Au-F127 nanospheres. Here, the Au-F127 nanospheres could provide a blood compatible surface with antifouling property for determination of glucose in whole blood. The cyclic voltammetric results indicated that GOx immobilized on the Au-F127 nanospheres exhibited direct electron transfer reaction, and the cyclic voltammogram (CV) displayed a pair of well-defined and nearly symmetric redox peaks with a formal potential of 93 mV. The biosensor had good electrocatalytic activity toward glucose with a low detection limit 3.15 pM. The glucose biosensor did not respond to ascorbic acid (AA) and uric acid (UA) at their high concentration encountered in blood. In this method, the biosensor was used for quantification of the concentration of glucose in whole blood samples. The data obtained from the biosensor showed good agreement with those from a biochemical analyzer in hospital.