A highly sensitive label-free assay for the determination of blood coagulation Factor XIII activity is demonstrated through the controlled assembly of peptide-functionalized gold nanoparticles (AuNPs). Activated Factor XIII catalyzes the formation of covalent crosslinking between peptide chains through ε-(γ-glutamyl)-lysine bonds leading to the aggregation of the AuNPs and consequently a red-shift of the localized surface plasmon resonance. The selective engineering of nanoscale order over AuNP crosslinking via the formation of isopeptide bonds provides a new approach toward the design of nanoassemblies with precise control on the molecular level. The colorimetric assay reported here provides direct qualitative and quantitative analysis of Factor XIII activity with a limit of detection of 0.01 U mL(-1).