Nanoparticles(NPs)with a protein corona has attracted increasing interest due to itssignificance on nano-bio interface.NP-protein conjugates are promising probes for biologicaldiagnostics as well as versatile building blocks for nanotechnology.Here we demonstrate a facilemethod to prepare nanoparticles bearing discrete numbers of proteins(such as bovine serumalbumin,BSA)simply by physical adsorption and electrophoretic isolation,in which the proteinspecific amphiphilic properties play important roles and the number of adsorbed proteins can alsobe manipulated by tuning the coating extent of nanoparticles by the amphiphilic polymer.Inaddition,we investigated the binding site of the robust BSA corona formed on amphiphilicpolymer coated gold nanoparticles(AP-AuNPs)and discussed the possible mechanism.We haveidentified a 105-amino-acid peptide(12.2 kDa)as the "epitope" responsible for the robustBSA-NP interactions,and further tested the peptide sequence dependence by comparing theaffinity difference between two-epitope peptides and one non-epitope peptide.With thephotoluminescent amino acid residues,the fluorescence quenching method based on thenanometal surface energy transfer(NSET)principle was able to study the thermodynamics of thecurrent binding system.Finally,we proposed a mechanism that the robust protein-NP interaction formed by a mortise-tenon joint,in which the epitope peptide acted as the "tenon tongue" to insertinto "mortise holes" formed in the organic molecular layers on AP-AuNPs.These findings mightshed light on a new strategy for studying interactions between proteins and NPs,and further guidethe rational design of NPs for safe and effective biomedical applications.