Nanometer-sized fluorescent hybrid silica (NFHS) particles were prepared for use as sensitive and photostable fluorescent probes in biological staining and diagnostics. The first step of the synthesis involves the covalent modification of 3-aminopropyltrimethoxysilane with an organic fluorophore, such as fluorescein isothiocyanate, under N₂ atmosphere for getting a fluorescent silica precursor. Then the NFHS particles, with a diameter of well below 40 nm, were prepared by controlled hydrolysis of the fluorescent silica precursor with tetramethoxysilane (TMOS) using the reverse micelle technique. The fluorophores are dispersed homogeneously in the silica network of the NFHS particles and well protected from the environmental oxygen. Furthermore, since the fluorophores are covalently bound to the silica network, there is no migration, aggregation and leakage of the fluorophores. In comparison with common single organic fluorophores, these particle probes are brighter, more stable against photobleaching and do not suffer from intermittent on/off light emission (blinking). We have used these newly developed NFHS particles as a fluorescent marker to label antibodies, using silica immobilization method, for the immunoassay of human α-fetoprotein (AFP). The detection limit of this method was down to 0.05 ng mL⁻¹ under our current experimental conditions. We think this material would attract much attention and be applied widely in biotechnology.