In this study, we present an ultrasensitive sensor based on nanoporous anodic alumina (NAA) for detection of gold(III) ions (Au(3+) ions) using reflectometric interference spectroscopy (RIfS). Nanoporous anodic alumina, prepared by two-step electrochemical anodization, was functionalized with 3-mercaptopropyl-tirethoxysilane (MPTES) in order to selectively detect Au(3+) ions. Thus prepared, MPTES-NAA sensors were exposed to different concentrations of Au(3+) ions ranging from 0.1 to 750 μM and the changes in the effective optical thickness (ΔEOT) were monitored in real-time. The linear range of these Au(3+) sensors was from 0.1 to 80 μM, with a lower detection limit of 0.1 μM of Au(3+) ions. Furthermore, the specificity of these MPTES-NAA sensors was validated by sequential exposure to 40 μM solutions of Fe(3+,) Mg(2+), Co(2+), Cu(2+), Ni(2+), Ag(+), and Pb(2+), resulting in negligible changes in EOT as compared to the same concentration of Au(3+) ions. Detection of Au(3+) ions in complex and environmentally and biologically relevant solvents such as tap water and phosphate buffer solution (PBS) was also successfully carried out in order to demonstrate the real-life application of these sensors. Finally, the binding isotherm for Au(3+) ions and thiol (SH) group of MPTES-NAA system was determined by fitting the changes in EOT to Freundlich and Langmuir isotherm models.