Three-dimensionally structured, silica based, organic-inorganic hybrid nanoparticles (NPs) were prepared by a simple and feasible water-in-oil (W/O) microemulsion method and a promising platform for bioelectrochemical analysis was obtained. The commonly used phenathiazine organic compound, toluidine blue (TB) was readily captured in the three-dimensional cage of the inorganic SiO(2) network, which was considered to serve as a protective "shell" toward the embedded TB. A TEM image indicated the size of the thus prepared TB-doped SiO(2) (TB@SiO(2)) NPs was 21 +/- 3 nm. UV-visible and IR spectroscopy confirmed successful formation of the organic-inorganic composite and possible interaction between TB and SiO(2), which favored enhanced stability of the hybrid. A sensitive amperometric sensor for hemoglobin (Hb) biomolecules based on TB@SiO(2) NPs conjugated with a biopolymer chitosan (CHIT) membrane was then developed. The surface of the silica NPs was highly biocompatible and the TB captured inside maintained its high electron-transfer efficiency. Dye leakage of TB from the TB@SiO(2) hybrid was proved to be minimal, owing to the inorganic SiO(2) network and the force of interaction between TB and SiO(2). The amperometric sensor had a detection limit of 2.5 x 10(-9) mol L(-1) (S/N = 3) with a linear range from 5.0 x 10(-9) to 3.0 x 10(-6) mol L(-1) for Hb. When it was applied to determine the concentration of a clinical blood sample, satisfactory results were obtained which were in good agreement with those obtained by the standard method.