The ultimate nature of streptavidin to bind biotin tightly is widely utilized in many solid-phase based applications to provide a universal binding surface for biotinylated molecules. However, the preparation of the streptavidin coatings by passive adsorption may heavily alter the binding properties of native streptavidin and may not result in the best possible capture surface for demanding solid-phase assays. By introducing sulphydryl groups through primary amines in the protein, we have activated and conjugated native streptavidin into larger protein polymers resulting in high local binding density when coated on polystyrene. This thiolated streptavidin formed through chemical modification has improved adsorption properties and biotin binding capability, compared to the native streptavidin. When this thiolated streptavidin is coated on polystyrene, a dense surface is formed, which provides up to 3-fold increase in the biotin binding efficiency and improves the surface stability by minimizing the desorption of the adsorbed protein from the surface during incubation. Furthermore, this high-capacity surface is resistant to harsh chemical treatments, such as denaturing conditions or mild reducing conditions. The improved adsorption properties of the thiolated streptavidin allow the coating process to be performed with shorter incubation times (15min), still providing enhanced solid-phase properties, compared to a reference streptavidin surface.