DNA can be utilized as a generic delivery vector as well as a traditional biological material for DNA vaccination. Although the use of DNA as an antigen expression vector or a vaccine adjuvant has been intensively studied for several decades, the use of DNA molecules as a delivery carrier has not been explored until recently. This issue is probably due to the topological limitation of DNA in its natural linear or circular structure form. Multivalent DNA-based vector delivery platforms overcome this structural barrier and are particularly suited for DNA vaccine delivery because of their multifunctionality, monodispersity, anisotropicity, and bioconjugation ability with numerous functional moieties. In this chapter, we mainly describe the construction of multivalent DNA-based delivery vectors using DNA engineering methods. Specifically, the synthesis strategies for highly branched dendrimer-like DNA structures in general and methods for their application to DNA vaccine delivery are introduced.