DNA nanostructures generated through rolling circle amplification (RCA) technology have found widespread application in drug delivery systems due to their simple fabrication, modification, and unique structures for loading various types of drugs. In this chapter, we describe methods for generating DNA nanostructures using RCA technology and providing functionality with DNA aptamers. These methods involve non-covalent functionalization of DNA nanostructures with DNA aptamers. Methods for constructing DNA nanostructures, such as DNA nanoballs and DNA nanogels, are described with a focus on using RCA technology, as well as characterization of the nanostructures. Methods for the functionalization of DNA nanostructures include complementary hybridization of base pairs and the decoration of DNA nanostructures with a polyadenine-tailed DNA aptamer based on hydrophobic interactions. Methods for testing the modification of nanostructures with aptamers are described, with examples of experimental data provided. Consideration points for constructing the nanostructures and modifying them with DNA aptamers are noted. Methods for designing and characterizing DNA nanostructures modified with DNA aptamers will be useful for diverse biomedical applications such as targeted drug delivery and imaging for the treatment and diagnosis of diseases.