Quantitative research on the localized surface plasmon resonance of metallic nanostructures has received tremendous interest. However, most current studies have concentrated on theoretical calculation because it is difficult to obtain monodisperse metallic nanostructures with high purity experimentally. In this work, gold nanodimers (GNDs) with high uniformity are fabricated through DNA-directed self-assembly of gold nanoparticles and then used as substrates for surface-enhanced Raman scattering (SERS). The dependence of the SERS properties of the substrates on the size of the gold particles and the position of Rhodamine B (RB) molecules is systematically studied using the DNA-directed self-assembled GNDs with Raman-active RB molecules immobilized in different positions between pairs of gold nanoparticles as a model system. Theoretical simulations conducted using the finite difference time domain (FDTD) method confirm the structure-dependent SERS characteristics of the GNDs, with good agreement between the FDTD simulation data and experimental results.