Selective transport in nanochannels (protein-based ion channels) is already used in living systems for electrical signaling in nerves and muscles, and this natural behavior is being approached for the application of biomimetic nanochannels in biosensors. On the basis of this principle, single nanochannels and nanochannel arrays seem to bring new advantages for biosensor development and applications. The purpose of this review is to provide a general comprehensive and critical overview on the latest trends in the development of nanochannel-based biosensing systems. A detailed description and discussion of representative and recent works covering the main nanochannel fabrication techniques, nanoporous material characterizations, and especially their application in both electrochemical and optical sensing systems is given. The state-of-the-art of the developed technology may open the way to new advances in the integration of nanochannels with (bio)molecules and synthetic receptors for the development of novel biodetection systems that can be extended to many other applications with interest for clinical analysis, safety, and security as well as environmental and other industrial studies and applications.