Aptamer biosensors are one of the most powerful techniques in biosensing. Achieving the best platform to use in aptamer biosensors typically includes crucial chemical modifications that enable aptamer immobilization on the surface in the most efficient manner. These chemical modifications must be well defined. In this work we propose nanoporous anodic alumina (NAA) chemically modified with streptavidin as a platform for aptamer immobilization. The immobilization of biotinylated thrombin binding aptamer (TBA) was monitored in real time by means of reflective interferometric spectroscopy (RIfS). The study has permitted to characterize in real time the path to immobilize TBA on the inner pore walls of NAA. Furthermore, this study provides an accurate label-free method to detect thrombin in real-time with high affinity and specificity.