Since the introduction of DNA-based architectures, in the past decade, DNA tetrahedrons have aroused great interest. Applications of such nanostructures require structural control, especially in the perspective of their possible functionalities. In this work, an integrated approach for structural characterization of a tetrahedron structure is proposed with a focus on the fundamental biophysical aspects driving the assembly process. To address such an issue, spin-labeled DNA sequences are chemically synthesized, self-assembled, and then analyzed by Continuous-Wave (CW) and pulsed Electron Paramagnetic Resonance (EPR) spectroscopy. Interspin distance measurements based on PELDOR/DEER techniques combined with molecular dynamics (MD) thus revealed unexpected dynamic heterogeneity and flexibility of the assembled structures. The observation of flexibility in these ordered 3D structures demonstrates the sensitivity of this approach and its effectiveness in accessing the main dynamic and structural features with unprecedented resolution.