Reflective Interference Spectroscopy (RIfS) technology based on porous thin film as a sensor substrate provides many advantages for biomedical molecular characterization, among which detection sensitivity is crucial. Nanoporous Anodic Aluminum (NpAA), as an effective nanostructured sensor platform, combined with RIfS technology, has practical applications in studying biomolecular properties and interactions between drug molecules and biomolecules. However, challenges will be faced, when the sample quantity is small and the refractive index of solution changes slightly, which leading to issues such as weak intensity of RIfS spectral signal, insufficient detection sensitivity, and inaccurate in judgment results. In order to improve the practicality of the reflective interference spectroscopy based on nanoporous thin films in the detection of trace samples, a model is established in this study, and various conditions affecting sensitivity and mechanisms are quantitatively investigated and analyzed by combining experiment and simulation.First, two types of NpAA sensor substrates with same pore diameter (80 nm) but different pore depths (9 μm and 11 μm) were prepared with the standard two-step anodization method with oxalic acid as the electrolyte. The morphology of NpAA was characterised with scanning electron microscope, revealing well-ordered pore structures with vertical and distinct pore walls. The material has a high porosity, with significant gaps between the pores and a periodic arrangement. This pore structure enhances the film substrate's light field capture, creating standing wave phenomenon and significantly boosting the sensing signal strength. Different concentrations of glycerol solutions, with refractive indices ranging from 1.33 to 1.60, were then applied to the two types of NpAA sensor substrates. After the solution fully penetrated into the pores, the measurement was performed. The measurement data indicated that the sensitivity of RIfS for detecting trace amounts of filling liquid is 5.60×103 nm/RIU for a 9 μm depth and 8.00×103 nm/RIU for an 11 μm depth. In porous media, as the pore depth and porosity increase, much filling liquid will be in the pores. Generally, the effective refractive index of the porous film is influenced by both the refractive index of the bulk material and the filling material. For the same range of filling liquid refractive index changes, a greater porosity results in a larger effective refractive index changes, which in turn increases the effective optical thickness changes. Therefore, the sensitivity of nanoporous anodic alumina increases with the increase of pore depth, making the RIfS with NpAA as a sensing substrate more sensitive to changes in liquid refractive index.Next, a physical model of RIfS using nanoporous anodic alumina as a sensing substrate was established, and the impacts of pore depth and inner diameter on the RIfS sensing sensitivity were numerically investigated. The simulation results showed that the different pore depths correspond to different RIfS sensitivity, with the increase of pore depth, the sensitivity of RIfS is more sensitive to the change of the filling material inside the pores. For example, sensitivity increases from 2.20×103 nm/RIU to 8.30×103 nm/RIU as the filling liquid refractive index changes increasing from 0.22 (3 μm) to 0.06 (11 μm). Similarly, the RIfS sensitivity also varies with different inner diameters of NpAA films. As the inner diameter increases, the sensitivity increases from 2.80×103 nm/RIU with a filling liquid refractive index changes of 0.18 (30 nm) to 8.30×103 nm/RIU with a filling liquid refractive index changes of 0.06 (160 nm).The simulation data are highly consistent with the experimental results. The sensitivity simulation model and experimental results in this paper will promote the practical application of the RIfS technology based on nanoporous sensing substrate in the field of non-destructive and rapid analysis of molecule characteristics and their interaction properties at the single-molecule level.