Herein, we present a comparative study about the sensing performance of optical biosensors based on photoluminescence spectroscopy (PLS) and reflectometric interference spectroscopy (RIfS) combined with nanoporous anodic alumina (NAA) platforms when detecting different analytes under distinct adsorption conditions. First, NAA platforms are structurally engineered in order for optimizing the optical signals obtained by PLS and RIfS. Then, the most optimal NAA platforms combined with PLS and RIfS are quantitatively compared by detecting two different analytes: d-glucose and l-cysteine under nonspecific and specific adsorption conditions, respectively. The obtained results demonstrate that such parameters as the analyte nature and adsorption conditions play a direct role in the sensing performance of these platforms. However, as this study demonstrates, PLS-NAA platforms are more sensitive than RIfS-NAA ones. The former shows better linearity (i.e., proportional change in the sensing parameter with analyte concentration), higher sensitivity toward analytes (i.e., sharper change in the sensing parameter with analyte concentration), and lower limit of detection (i.e., minimum detectable concentration of analyte).