Porous silicon (PSi) is a promising biomaterial presenting the advantage of being biocompatible and bioresorbable. Due to the large specific surface area and unique optical features, these microporous structures are excellent candidates for biosensing applications. Investigating device functionality and developing simple Si-based transducers need to be addressed in novel biological detection. Our work demonstrates that, among the various PSi configurations for molecular detection, PSi microcavity structure demonstrates the best biosensing performance, reflected through the enhanced luminescence response and the changes in the refractive index. For successful immobilization, molecular infiltration and confinement are the two key factors that are controlled by the pore size distribution of the PSi microcavities and by the surface modification obtained by silane-glutaraldehyde chemistry. Enhancement of the fluorescence emission of confined fluorescent biomolecules in the active layer of PSi microcavities was observed for a nonlabeled protein with a natural green fluorescence, the glucose oxidase enzyme (GOX). An increase in the fluorescence emission was also observed when functionalized PSi material was used to detect specific binding between biotin and a low concentration of labeled streptavidin. Evidence for the enzymatic activity of GOX in its adsorbed form is also presented. Use of smart silicon devices, enabling enhancement of fluorescence emission of biomolecules, offers easy-to-use biosensing, based on the luminescence response of the molecules to be detected.