DNA origami technology allows for the precise nanoscale assembly of chemical entities that give rise to new functional materials. We have created a versatile DNA Origami Nanofork Antenna (DONA) by assembling Au or Ag nanoparticle dimers with 1.17 ± 0.67 nm gap size, enabling signal enhancements in surface-enhanced Raman scattering (SERS) of up to 1011. This allows for single-molecule SERS measurements, which can even be performed with larger gap sizes to accommodate differently sized molecules, and at various excitation wavelengths. A general scheme is presented to place single analyte molecules into the SERS hot spots using the DNA origami structure exploiting covalent and non-covalent coupling schemes. By using Au and Ag dimers, single-molecule SERS measurements of three dyes and cytochrome c and horseradish peroxidase proteins are demonstrated even under non-resonant excitation conditions, thus providing long photostability during time-series measurement, and enabling unprecedented optical monitoring of single molecules and DNA origami based nanomachines.