We synthesized gold nanoparticles at linoleic acid (GNPs@linoleic acid) for glucose sensing application with the help of fluorescence spectroscopy. The synthesis is a non-toxic process. So, the synthesized nanoparticles are safe and biologically compatible with the human body. The synthesis process uses a polyunsaturated fatty acid called linoleic acid that surrounds the GNPs and acts as a reducing and stabilizing agent. In this procedure, first, we achieve the optimal synthesis method by changing the gold salt concentration, which leads to obtaining the maximum sensitivity to changing the glucose concentration. For this purpose, we considered the optimal concentration of gold salt 2 mM. Eventually, gold nanoparticles remain in linoleic acid. In the next stage, we obtain the fluorescence spectra of the synthesized GNPs@linoleic acid (at the excitation wavelength of 405 nm) in the presence of different glucose concentrations. Our measurements show an increase in glucose concentration reduces the peak intensity of the fluorescence for the GNPs@linoleic acid. The main reasons for this reduction are the degradation and auto-oxidation of linoleic acid, catalytic features of GNPs, and the presence of GNPs@linoleic acid in measuring glucose concentrations, enhancing the sensitivity as compared to that of linoleic acid alone by 4.8 times. The synthesized GNPs@linoleic acid can measure the glucose concentration within the linear range of 0–300 mg<inline-formula> <tex-math notation="LaTeX">$\cdot $ </tex-math></inline-formula>dL<sup>−1</sup> (i.e., 0-16.65 mM) by a detection limit of 50 mg<inline-formula> <tex-math notation="LaTeX">$\cdot $ </tex-math></inline-formula>dL<sup>−1</sup> (~2.8 mM). This measurement range is suitable for clinical applications — i.e., within 70 to 120 mg<inline-formula> <tex-math notation="LaTeX">$\cdot $ </tex-math></inline-formula>dL<sup>−1</sup>.