We developed a new quantitative detection method for an immunoreaction combining magnetite (Fe₃O₄) nanoparticle labeling with confocal Raman spectrometry. Magnetite nanoparticles are transparent, Raman active, and easily can be controlled easily by magnets in bimolecular reactions. Human Chorionic Gonadtropin (hCG) was selected as a target molecule for this investigation. Sandwich immunoreaction was performed on a dot-patterned substrate using two anti-hCG antibodies, which were the antibody immobilized on the substrate and the biotinylated antibody. The immunoreaction was microscopically visualized by reacting streptavidin-modified magnetite nanoparticles with the biotinylated antibody on the substrate. The adsorbed magnetite nanoparticles on the dot pattern were detected by Raman intensity imaging. A good linear relation between the integrated Raman intensity spectra at 220 cm^-1 and hCG concentration was gained. This method may be applied toward the detection of other target molecules in the fields of biotechnology and biomedicine. [DOI: 10.1380/ejssnt.2008.142]