afforded by nanoscale technologies are expected to have substantial impacts on almost all industries and areas of society (e.g., medicine, plastics, energy, electronics, and aerospace) [1][2][3][4][5]. The recent advancement of bioanalytical techniques involving the development of highly sensitive or selective analytical methods based on nanoscale materials/molecules have progressed with remarkable success. In this regard, silver nanoparticles (AgNPs) have received significant attention in constructing analytical and bioanalytical sensors. This relevance arises from their unusual optical, electronic, and chemical properties [6][7][8]. They have a high surface area, very small size (<20 nm) and high dispersion ability, spectrally selective coating [9, 10], surface-enhanced Raman scattering [11,12], and antibacterial activity [13]. Moreover, AgNPs exhibit optical properties which could not be observed either in molecular or in bulk metallic form [14][15][16][17].Fluorescence spectroscopy as a sensitive, simple, diverse, and nondestructive method can provide real time, in situ, and dynamic analytical information [18]. However, utilizing nanoparticles in fluorescence analysis has provided significant improvement in this area [19][20][21][22][23]. It should be mentioned that the fluorescence of metal clusters and thin films is well established based on Mooradian's observation of photoluminescence from bulk copper and gold [24]. Also several reports are available related to the fluorescence of silver nanoclusters [25,26].Glucose, a simple sugar (monosaccharide), is an important carbohydrate in biology. Cells use it as a source of energy and a metabolic intermediate and its lack or excess can produce detrimental influence on cellular functions. The glucose level in blood is considered as a clinical indicator for diabetes. Therefore, the monitoring of glucose level in blood with faster and more accurate methods is in great demand and a great deal of effort has been focused Abstract A nanosensor was designed for the enzymatic determination of glucose based on the fluorescence enhancement of silver nanoparticles (AgNPs). This enhancement is the result of the glucose oxidase-catalyzed oxidation of glucose. Silver nanoparticles were prepared through a chemical reduction by using trisodium citrate as the reducing agent and were characterized with UV-visible spectroscopy, Fourier transform infrared spectroscopy and transmission electron microscopy. The experimental results showed that the increase of the AgNP fluorescence was linearly proportional to the concentration of glucose within its concentration ranges of 2.50 × 10 −5 -7.50 × 10 −3 M and 7.50 × 10 −3 -7.50 × 10 −2 M with a detection limit of 5.53 × 10 −7 M under the optimized experimental conditions. To evaluate the applicability of the nanobiosensor, determination of glucose in real samples was performed according to the developed procedure.