The past decade witnessed the fast evolvement of structural DNA nanotechnology, which uses DNA as blueprint and building material to construct artificial nanostructures. Using branched DNA as the main building block (also known as a "tile") and cohesive single-stranded DNA (ssDNA) ends to designate the pairing strategy for tile-tile recognition, one can rationally design and assemble complicated nanoarchitectures from specifically designed DNA oligonucleotides. Objects in both two- and three-dimensions with a large variety of geometries and topologies have been built from DNA with excellent yield; this development enables the construction of DNA-based nanodevices and DNA-template directed organization of other molecular species. The construction of such nanoscale objects constitutes the basis of DNA nanotechnology. This chapter describes the protocol for the preparation of ssDNA as starting material, the self-assembly of DNA nanostructures, and some of the most commonly used methods to characterize the self-assembled DNA nanostructures.