During the last decades, by virtue of their unique physicochemical properties and potential application in microelectronics, biosensing and biomedicine, metal nanomaterials (MNs) have attracted great research interest and been highly developed. Deoxyribonucleic acid (DNA) is a particularly interesting ligand for templating bottom-up nanopreparation, by virtue of its excellent properties including nanosized geometry structure, programmable and artificial synthesis, DNA-metal ion interaction and powerful molecular recognition. DNA-templated copper nanoparticles (DNA-CuNPs) has been developed in recent years. Because of its advantages including simple and rapid preparation, high efficiency, MegaStokes shifting and low biological toxicity, DNA-CuNPs has been highly exploited for biochemical sensing from 2010, especially as a label-free detection manner, holding advantages in multiple analytical technologies including fluorescence, electrochemistry, surface plasmon resonance, inductively coupled plasma mass spectrometry and surface enhanced Raman spectroscopy. This review comprehensively tracks the preparation of DNA-CuNPs and its application in biosensing, and highlights the potential development and challenges regarding this field, aiming to promote the advance of this fertile research area.