The control of the assembly of nanostructures of dye-coded oligonucleotide silver nanoparticle (DOSN) conjugates (silver nanoparticles cofunctionalized with thiol modified DNA and a Raman reporter molecule) in a sandwich assay format with single base discrimination in an unmodified target oligonucleotide is reported. Rational placement of a single base mismatch in the 18 base duplex formed between target and DOSN conjugates completely controls the hybridization event, preventing or allowing nanostructure formation. The assembly process was monitored using UV-vis spectrometry and surface-enhanced resonance Raman scattering (SERRS). These two supporting analysis techniques show that there is an initial formation of small, highly SERRS active assemblies followed by the formation of larger superaggregates. The rapid increase in SERRS intensity obtained from DOSN conjugate hybridization has been utilized for the near-immediate discrimination of a single base mismatch using SERRS. This report highlights the exquisite control and detection possibilities offered by coupling specific molecular recognition events, such as DNA hybridization, with the optical and SERRS responses possible by nanoparticle assembly.