In this study, we report about the structural engineering and optical optimization of nanoporous anodic alumina rugate filters (NAA-RFs) for real-time and label-free biosensing applications. Structurally engineered NAA-RFs are combined with reflection spectroscopy (RfS) in order to develop a biosensing system based on the position shift of the characteristic peak in the reflection spectrum of NAA-RFs (Δλpeak). This system is optimized and assessed by measuring shifts in the characteristic peak position produced by small changes in the effective medium (i.e., refractive index). To this end, NAA-RFs are filled with different solutions of d-glucose, and the Δλpeak is measured in real time by RfS. These results are validated by a theoretical model (i.e., the Looyenga-Landau-Lifshitz model), demonstrating that the control over the nanoporous structure makes it possible to optimize optical signals in RfS for sensing purposes. The linear range of these optical sensors ranges from 0.01 to 1.00 M, with a low detection limit of 0.01 M of d-glucose (i.e., 1.80 ppm), a sensitivity of 4.93 nm M(-1) (i.e., 164 nm per refractive index units), and a linearity of 0.998. This proof-of-concept study demonstrates that the proposed system combining NAA-RFs with RfS has outstanding capabilities to develop ultrasensitive, portable, and cost-competitive optical sensors.