A single-step gold nanoparticle (AuNP)-based immunoassay is demonstrated in which the nanoparticle surface is tagged with short viral peptide epitopes. Antiviral antibodies with monoclonal specificity trigger nanoparticle aggregation yielding a colorimetric response that enables detection of antibodies in the low-nanomolar range within a few minutes. In silico insights into the interactions at the epitope–gold interface demonstrate that the conformational landscape exhibited by the epitopes is strongly influenced by the amino acid sequence and location of particular residues within the peptides. The conformation, orientation, and linker chemistry of the peptides affect the immune complex formation in nonintuitive ways that are, nevertheless, explained by a unique sterically kinetically driven aggregation mechanism. The rapid and specific performance of the AuNP immunoassay may have generic potential in point of care diagnostics and other laboratory routines.