Graphite nanoparticle (GN), consisting of several stacked graphene sheets, shows homogenous spherical shape, high conductivity, and large surface area for biomolecular conjugation. We utilized these unique characteristics of GN for constructing an enzyme biosensor to detect glucose in real samples. To immobilize the glucose oxidase (GOx) on the GN, the surface of the GN was first modified by 1-pyrenebutyric acid N-hydroxysuccinimide ester (Py-NHS ester) by pi-pi stacking interaction between the pyrene moiety and the GN to produce the GN-Py-NHS ester conjugates, and then the GOx was linked with GN-Py-NHS ester through amide bond. The resultant GN-Py-GOx composite was mixed with a Nafion solution and deposited on the glassy carbon electrode as a thin film, and used for direct electron transfer-based glucose sensing. Amperometric response linearly increased in proportional to the glucose concentration up to 2.2 mM, and the detection limit was measured as 50 mu M with sensitivity of 7.29 x 10(-2) nA mM(-1) cm(-2). The spiked glucose in a real urine sample was also successfully analyzed, and the GN-Py-GOx biosensor performance was maintained for one month without loss of activity and sensitivity.