A multicomponent nanostructure comprising of gold nanorod–nanoparticle (AuNR–AuNP) composites was fabricated to detect thrombin at subnanomolar concentrations in diluted human blood serum. Simulation and experiments revealed that the strong electromagnetic coupling resonance at the nanorod–nanoparticle junction of these probes can be used to construct highly sensitive SERS aptasensors.