With the development of nanotechnology, great progress has been made in the fabrication of nanochannels. Nanofluidic biochips based on nanochannel structures allow biomolecule transport, bioseparation, and biodetection. The domain applications of nanofluidic biochips with nanochannels are DNA stretching and separation. In this Review, the general fabrication methods for nanochannel structures and their applications in DNA analysis are discussed. These representative fabrication approaches include conventional photolithography, interference lithography, electron-beam lithography, nanoimprint lithography and polymer nanochannels. Other nanofabrication methods used to fabricate unique nanochannels, including sub-10-nm nanochannels, single nanochannels, and vertical nanochannels, are also mentioned. These nanofabrication methods provide an effective way to form nanoscale channel structures for nanofluidics and biosensor devices for DNA separation, detection, and sensing. The broad applications of nanochannels and future perspectives are also discussed.