Surface-enhanced Raman scattering (SERS)-based sensing is promising in that it has potential to allow for highly sensitive, selective, and multiplexed detection and imaging. However, the controlled assembly and gap formation between plasmonic particles for generating strong SERS signals in a quantitative manner is highly challenging, especially on biodetection platforms, and particle-to-particle variation in the signal enhancement can vary by several orders of magnitude in a single batch, largely limiting the reliable use of SERS for practical sensing applications. Here, a hierarchic-nanocube-assembly based SERS (H-Cube-SERS) bioassay to controllably amplify the electromagnetic field between gold nanocubes (AuNCs) is developed. Based on this strategy, H-Cube-SERS assay allows for detecting target DNA with a wide dynamic range from 100 aM to 10 pM concentrations in a stable and reproducible manner. It is also found that the uniformly formed AuNCs with flat surfaces are much more suitable for highly sensitive, reliable, and quantitative biodetection assays due to faster DNA binding kinetics, sharper DNA melting transition, wider hot spot regions, and less dependence on light polarization direction than spherical Au nanoparticles with curved interfaces. This work paves the pathways to the quantitative and sensitive biodetection on a SERS platform and can be extended to other particle assembly systems.