Luminescent ruthenium(II) complex covalently bound silica nanoparticles have been prepared and used as a probe for time-gated luminescence bioimaging. The new nanoparticles were prepared by copolymerization of a luminescent Ru(II) complex tris(5-amino-1,10-phenanthroline)ruthenium(II) conjugated with 3-aminopropyl(triethoxy)silane (APS-Ru conjugate), free (3-aminopropyl)triethoxysilane (APS) and tetraethyl orthosilicate (TEOS) in a water-in-oil reverse microemulsion consisting of Triton X-100, n-octanol, cyclohexane and water in the presence of aqueous ammonia. Characterization by transmission electron microscopy indicates that the nanoparticles are monodisperse, spherical and uniform in size, 64 ± 4 nm in diameter. Compared with the dye-doping nanoparticles, dye leakage of the new nanoparticles was remarkably decreased. In addition, it was found that the Ru(II) complex luminescence could be effectively enhanced with a longer luminescence lifetime (∼2.3 μs) after forming the nanoparticles, which enables the nanoparticles to be suitable as a bioprobe for time-gated luminescence bioimaging applications. The nanoparticle-labeled streptavidin was prepared and successfully used for time-gated luminescence imaging detection of an environmental pathogen, Giardia lamblia, with high specificity and sensitivity.