Here, the results of a study of the mechanism of bio-enabled surface-mediated titania nanoparticle synthesis with assistance of polyelectrolyte surfaces are reported. By applying atomic force microscopy, surface force spectroscopy, circular dichroism, and in situ attenuated total reflection Fourier-transform infrared spectroscopy, structural changes of rSilC-silaffin upon its adsorption to polyelectrolyte surfaces prior to and during titania nanoparticle growth are revealed. It is demonstrated that the adhesion of rSilC-silaffin onto polyelectrolyte surfaces results in its reorganization from a random-coil conformation in solution into a mixed secondary structure with both random coil and β-sheet structures presented. Moreover, the protein forms a continuous molecularly thin layer with well-defined monodisperse nanodomains of lateral dimensions below 20 nm. It is also shown that rSilC embedded inside the polylelectrolyte matrix preserves its titania formation activity. It is suggested that the surface-mediated, bio-enabled synthesis of nanostructured materials might be useful to develop general procedures for controlled growth of inorganic nanomaterials on reactive organic surfaces, which opens new perspectives in the design of tailored, in situ grown, hybrid inorganic–organic nanomaterials.