Tunable fluorescent silica nanoparticles (SiO₂ NPs) with variable sizes based on fluorescence resonance energy transfer were synthesized. The SiO₂ NPs were obtained by the Störber method by varying tetraethyl orthosilicate (TEOS) concentrations and with the incorporation of different concentrations of fluorescein (Fl) and rhodamine B (RhB) conjugated with 3-(aminopropyl)triethoxysilane. We thus recorded homogeneous SiO₂ NPs of different sizes. By transmission electron microscopy imaging, the sizes of 200, 280, and 380 nm were determined. The Fl and RhB fluorescent dyes showed well-overlapped emission from the fluorescent energy donor to the energy acceptor with the optimal ratio of quantum yields. By static fluorescence, varied emissions were recorded according to the concentration ratio of the donor–acceptor pair. Increasing intensity values were collected with the addition of higher concentrations of the fluorescent energy donor and decreased fluorescent lifetime decays. By laser fluorescence microscopy, higher enhanced surfaces were produced in the presence of both emitters, as compared with those in the presence of mono-colored SiO₂ NPs with optimal excitation with the fluorescent energy donor only. These NPs were well dispersed in polar solvents; however, due to their polar surface and size, higher interactions produced dimeric nanoaggregates. In addition, to evaluate their applications, their depositions were evaluated over modified glass slide substrates for smart light-responsive materials and over Cyanobacteria and Escherichia coli for the development of nanobiostructures with varied optical activities.