Spherical Ru(bpy)(2+)(3)-doped silica (RuSi) nanoparticles were prepared via a water-in-oil microemulsion approach. The electrochemical and electrochemiluminescent properties of the RuSi nanoparticles immobilized on an indium tin oxide (ITO) electrode were investigated. Further, electrochemiluminescence (ECL) of the RuSi nanoparticles with covalently coated biomacromolecules was studied. By covalent cross-linking with glutaraldehyde, gamma-(aminopropyl) triethoxysilane (APTES)-pretreated RuSi nanoparticles were coupled with different concentrations of bovine serum albumin (BSA), hemoglobin, and myoglobin, respectively. ECL from these biomacromolecule-coated RuSi nanoparticles decreased with the increase of the loading of biomacromolecules. Moreover, the ECL of coreactants with different sizes was studied. The ECL decrease could be assigned to the steric hindrance and limited diffusion of coreactant molecules into the RuSi nanoparticles after biomacromolecule conjugation. Since tens of thousands of Ru(bpy)(2+)(3) molecules are contained in the silica particles and the RuSi nanoparticle surface modification could improve their biocompatibility, the biomacromolecule-coated RuSi nanoparticles could be readily used as efficient and stable ECL tag materials in the future.