A simple fluorescent sensing of glucose in aqueous fluids (e.g. tear fluid) using dually functionalized gold nanoparticles is presented. As a first step gold nanoparticles (AuNPs) were synthesized using oxidised dextran which acted both as reducing and stabilizing agent. Aminophenyl boronic acid was conjugated onto AuNPs by Schiff's base formation and the formed Schiff's base was stabilized by sodium borohydride reduction. Rhodamine B isothiocyanate (RBITC) was then assembled onto the modified AuNPs. The fluorescence of RBITC was nearly quenched and found to be revived when glucose was added. It is reasoned that the glucose binding induces restructuring of the surface assembly resulting in an overall increase in the size and thereby enhancing the distance between the gold core and fluorophore. TEM image and size measurements using dynamic light scattering (DLS) in fact, reflected this possibility. The increase in fluorescence was proportional with the concentration of glucose enabling quantitative detection. A good linearity was observed between the fluorescence intensity and glucose concentration in a range of 0.025–0.125μM with detection limit of 0.005±0.002μM. The potential of the method was demonstrated by measuring glucose in real tear fluids collected from volunteers. The method is extremely sensitive and can be employed to measure low concentration of glucose in aqueous fluids such as tear.