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A new type of Cy5-encapsulated photostable fluorescent silica nanoparticles (FSNPs) bearing positive charges have been successfully fabricated by a reverse microemulsion synthesis in one-pot. The Cy5 dye containing four primary amines are embedded into silica via covalent bonds through a silane coupling agent (GPTMS), followed by co-condensation with tetraethylorthosilicate. The uniform-sized, spherical and monodispersed FSNPs have high fluorescence intensity and photostability. The FSNPs exhibit high stability, good biocompatibility as well as low cytotoxicity. These FSNPs can be internalized into live cells and thus fluorescently label the cells. This study provides a simple synthesis approach that can be applied to other water-soluble and amino-modified organic dye molecules for biological targeting and fluorescent cell imaging.