DNA　not　only　plays　a　vital　role　in　nature　as　fundamental　hereditary　material　for　storing　genetic　material,　but　also　serves　as　well-defined　functional　material,　for　example,　building　blocks　for　the　assembly　of　nanoscale　bio-architectures　by　Watson-Crick　base-pairing　interaction.　With　the　development　of　molecular　biology,　biotechnology　and　nanoscience,　structural　DNA　nanotechnology　has　achieved　numerous　advances,　contributing　to　the　construction　of　various　DNA　nanostructures　ranging　from　discrete　objects　to　one　dimensional　(1D),　two　dimensional　(2D),　and　three　dimensional　(3D)　architectures.　Among　them,　DNA　tetrahedral　nanoarchitecture　is　intensively　studied　because　of　simple　3D　structure,　easy　design　and　unique　properties,　such　as　high　rigidity,　desirable　biostability　and　efficient　cellular　uptake　without　auxiliary　species.　This　review　summarizes　the　research　progress　in　the　assembly　of　DNA　tetrahedral　objects　and　outlines　the　applications　in　biosensing,　drug　delivery　and　targeted　therapy.　Moreover,　the　dependence　of　biological　activity　of　biomolecules　on　DNA　tetrahedron-mediated　spatially-controlled　arrangement　and　great　potential　applications　are　discussed.　In　addition,　the　challenges　in　the　design　and　clinic　applications　of　DNA　tetrahedron-based　platforms　are　described,　the　perspectives　towards　biomedical　applications　are　foreseen,　and　our　understandings　on　further　studies　of　DNA　tetrahedron　are　provided,　aiming　to　motivate　the　development　of　DNA　nanotechnology　and　interdisciplinary　research.　©　2023　The　Authors