We have developed a simple method to prepare bright and photostable luminescent silica nanoparticles of different sizes and narrow size distribution in high yield. The method is based on the use of Stober synthesis in the presence of a fluorophore to form bright silica nanoparticles. Unlike micro-emulsion-based methods often used to prepare luminescent silica particles, the Stober method is a one-pot synthesis that is carried out at room temperature under alkaline conditions in ethanol:water mixtures and avoids the use of potentially toxic organic solvents and surfactants. Our luminescent particles contained the transition metal complex tris(1,10-phenanthroline) ruthenium(II) chloride, [Ru(phen)(3)]Cl-2. They showed higher photostability and a longer fluorescence lifetime compared to free Ru(phen)(3) solutions. Leakage of dye molecules from the silica particles was negligible, which was attributed to strong electrostatic attractions between the positively charged ruthenium complex and the negatively charged silica. To demonstrate the utility of the highly luminescent silica nanoparticles in bioassays, we further modified their surface with streptavidin and demonstrated their binding to biotinylated glass slides. The study showed that digital counting of the luminescent nanoparticles could be used as an attractive alternative to detection techniques involving analogue luminescence detection in bioanalytical assays.