Polymer brushes have been widely used to functionalize surfaces and provide antifouling capabilities against proteins and cells. Many efforts have focused on methods for functionalization of antifouling polymer brush surfaces for interactions with specific cells, proteins and bacteria, but none have focused on immobilizing nanoparticles on these surfaces. This paper demonstrates that both pristine nanoparticles and protein-coated nanoparticles can adsorb onto well-functioning antifouling polymer brush coatings formed from poly-L-lysine-graft-poly(ethylene glycol) (PLL-g-PEG) and methoxy PEG-thiol. The role of ionic strength in solution, substrate surface material and nanoparticle surface charge on the interaction was investigated to explore the forces behind the interaction. The adsorption of different types of nanoparticles to the surface was studied, determining that both polystyrene, gold, carbon black and silica particles can adsorb onto PLL-g-PEG. We show that the approach can be applied in, and studied by, both surface plasmon resonance and fluorescence imaging and suggest its application as a means to study nanoparticle-protein interactions, such as the protein corona. Nanoparticles self-assembled at antifouling polymer brush surfaces provides a novel platform for both scientific studies and applications in biotechnology.