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DNA nanotweezers (DTs) are reversible DNA nanodevices that can optionally switch between opened and closed states. Due to their excellent flexibility and high programmability, they have been recognized as a promising platform for constructing a diversity of biosensors and logic gates, as well as a versatile tool for molecular biology studies. In this review, we provide an overview of biosensing applications using DTs. First, the design and working principle of DTs are introduced. Next, the signal producing principles of DTs are summarized. Furthermore, biosensing applications of DTs for varying targets and purposes, both in buffers and complex biological environments, are highlighted. Finally, we provide potential opportunities and challenges for the further development of DTs.