Nucleic acid hybridization is crucial in target recognition with respect to in vitro and in vivo nucleic acid biosensing. Conventional linear probes and molecular beacons encounter challenges in multiplexing and specific recognition of intractable nucleic acids. Advances in nucleic acid nanotechnologies have resulted in a set of novel structural probes: junction probes (JPs), which make full use of the advantages of specificity, stability, programmability and predictability of Watson-Crick base pairing. In recent years, junction probes have been regularly implemented in constructing systems related to biosensing, synthetic biology and gene regulation. Herein, we summarize the latest advances in JP designs as potential nucleic acid biosensing systems and their expansive applications, and provide some general guidelines for developing JP based sensing strategies for implementation of such systems.