Nanometer sized silica nanoparticles (SiO2-NP) were prepared in water and loaded with two organic compounds, namely perylene and 1,6-diphenyl-1,3,5-hexatriene, which have well-defined and known fluorescence properties. The size of void and dye-doped SiO2-NP were determined by both transmission electron microscopy and atomic force microscopy, which allowed determining the loading effects on the particle size and morphology. Differently loaded nanoparticles were characterized by both steady-state and time-resolved spectrofluorimetric techniques. The spectroscopic characterization allowed in the first place to establish where the dye molecules are localized within the particles and, later, to evaluate the sensing capability of the hybrid materials with respect to proteins. In particular, dye molecules resulted to have a bimodal distribution on the particle template, specifically (i) at the particle/water interphase and (ii) in close contact with the silica surface (in the inner particle). To prove the ability of the as-prepared and characterized particles to interact with proteins, BSA and RNA-si were used as models; the particle fluorescence was used as a sensitive tool to monitor the occurrence of such interactions. In all cases, proteins interact very efficiently with the SiO2-NP mainly through static interactions likely determined by electrostatic forces. A quantitative analysis of the steady-state fluorescence quenching experiments allowed to estimate the interaction radius, which is a useful parameter to sense and to discriminate proteins.