Tetrahedral framework nucleic acids (tFNAs) represent a promising advancement in nucleic acid nanotechnology due to their unique structural properties, high biocompatibility, and multifaceted biomedical applications. Constructed through a one-pot annealing method, four single-stranded DNAs self-assemble into stable, three-dimensional tetrahedral nanostructures with enhanced mechanical robustness and physiological stability, resisting enzymatic degradation. Their ability to permeate mammalian cells without transfection agents, coupled with modifiable surfaces, positions tFNAs as versatile carriers for drug and gene delivery systems. The tFNA-based platforms exhibit superior therapeutic efficacy, including antioxidative and anti-inflammatory effects, alongside efficient cellular uptake and tissue penetration. These features underpin their role in precision medicine, enabling targeted delivery of diverse therapeutic agents such as synthetic compounds, peptides, and nucleic acids. Additionally, tFNAs demonstrate significant potential in regenerative medicine, immune modulation, antibacterial strategies, and oncology. By addressing challenges in translational integration, tFNAs stand poised to accelerate the development of biomedical research and clinical applications, fostering novel therapies and enhancing therapeutic outcomes across a wide spectrum of diseases. This Perspective thoroughly details the unique attributes and diverse applications of tFNAs and critically evaluates tFNAs' clinical translational potential, outlining inherent implementation challenges and exploring potential solutions to these obstacles.