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 (NPs) on these surfaces. This article demonstrates that both pristine NPs and protein-coated NPs 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 NP surface charge in the interaction was investigated to explore the forces behind the interaction. The adsorption of different types of NPs onto the surface was studied, determining that 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 NP-protein interactions, such as the protein corona. NPs self-assembled at antifouling polymer brush surfaces provide a novel platform for both scientific studies and applications in biotechnology.