Surface-functionalized colloidal nanoparticles provide a versatile scaffold for protein surface recognition because of their size, the ability to tailor nanoparticle surfaces with a wide range of functionalities, and the ability to template the surface monolayer. Furthermore, the wide range of core materials such as TiO2 and Fe3O4 etc., enhance the utility of these particles. Owing to its high electron affinity for many proteins and enzymes, the dye Cibacron blue (CB) has been attached to various solid supports such as Sephadex, Sepharose, and polyacrylamide. The diversity shown by the blue dye in binding several unrelated classes of proteins makes it a potential candidate in terms of studies of the immobilized ligand. A facile method of synthesis and utility of a new, cheaper, and robust support material, TiO2 nanoparticles (NPs), functionalized with Cibacron blue dye for protein (lactic dehydrogenase) purification by affinity chromatography is described. The TiO2–CB nanoparticles do not lose their binding property even upon long storage, drying, and re-suspending into buffer multiple times. Studies carried out with Fe3O4–CB nanoparticles have also shown that they bind lactic dehydrogenase similar to TiO2–CB nanoparticles.