Recent advances in nanotechnology are mostly related to the development of newly engineered nanoparticles. New studies of biosensors modified with nanoparticles provide essential impetus for the treatment of diseases that adversely affect human health. Among these materials, Graphene (Gr), with very satisfactory electrical, chemical and physical features, is mainly used for the production of graphene based nano hybrids. In this paper a new surface with a great potential in biosensor applications is reported. For this purpose, a glassy carbon electrode surface was covered with a graphene (Gr)/gold nanoparticles (AuNPs)/nafion (Nf). The best performance and working conditions of the surface examined in detail and the surface morphology of the resulting film was evaluated by AFM, SEM, and surface contact angle analysis. The linear working range of a biosensor, a parameter of a particular importance for the glucose determination, was determined as range of 5 x 10(-6)-5 x 10(-4) M with 0.9706 R-2. It is observed that the prepared biosensor has an exceptionally low detection limit of 0.005 mu M for glucose. The effects of quantity of enzyme and glutaraldehyde on the response of glucose biosensor were elucidated. The pH and temperature effects on the response of the glucose biosensor were also investigated. Optimum pH and temperature were found as 8.0, 25 degrees C to be respectively. The K-m and V-max values constants, which are important kinetic constants for the enzymes, were also studied and found to be 0.0139 mM, 4.34 mu A min(-1) respectively. Repeatability and storage stability are significant parameters in biosensor studies. Results of 22 measurements showed that the biosensor maintained 82.6% of its initial activity. At the end of the 42nd day, the biosensor continued to have 74.79% of the initial amperometric response. The studies of storage stability of the biosensor revealed that Gr/AuNPs/Nf-GCE biosensors showed a remarkable sensitivity, wide linear detection ranges, low detection limits, and prolonged stabilities. There are studies reporting on graphene based biosensors. However, as far as of the knowledge of the authors the use of nafion with Gr/AuNPs on biosensor applications and also its effect of enzyme, glutaraldehyde, and storage stability has not been reported in the literature.