Anisotropic nanoparticles can provide considerable opportunities for assembly of nanomaterials with unique structures and properties. However, most reported anisotropic nanoparticles are either difficult to prepare or to functionalize. Here we report a facile one-step solution-based method to prepare anisotropic DNA-functionalized gold nanoparticles (a-DNA-AuNP) with 96% yield and with high DNA density (120 + 20 strands on the gold hemisphere). The method is based on the competition between a thiolated hydrophilic DNA and a thiolated hydrophobic phospholipid and has been applied to prepare a-DNA-AuNPs of different sizes and with a variety of DNA sequences. In addition, DNA strands on the a-DNA-AuNPs can be exchanged with other DNA strands with a different sequence. The anisotropic nature of the a-DNA-AuNPs allows regioselective hetero- and homonuclear assembly with high monodispersity, as well as regioselective functionalization of two different DNA strands for more diverse applications.