The production of uniform protein-binding biofunctional fluorescent spherical silica core-shell nanoparticles by a modified Stöber method is described. Fluorescent particle cores with diameters of 100 nm are synthesized in a two-step reaction. Functional shells for subsequent coupling reactions are provided by generating organic shells providing amine and carboxyl groups at the nanoparticles' shell surface. Conjugation of proteins to the nanoparticles is achieved using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) as coupling agent. The characterization of the nanoparticle systems and their surface functionalization is done by microelectrophoresis, dynamic light scattering (DLS), and a colorimetric detection of the amount of nanoparticle-attached protein via a bicinchoninic acid (BCA) assay. Fluorescently spiked nanoparticle cores with biofunctional shells for molecular recognition reactions may be used as imaging tools or reporter systems.