DNA network structures are used as a material with versatile uses and applications. Spherical nanostructure made of DNA, termed the DNA nanosphere, is one type of such DNA network structure, and it has a potential for applications in various areas of biological engineering such as drug delivery and gene therapy. This work presents a study of DNA nanospheres for applications in two different areas of bioengineering that are yet to be explored with DNA nanospheres: cell-free protein expression and dye tracing in biological organisms. For cell-free protein expression, we believed that DNA nanospheres would induce a macromolecular crowding environment around the genes, leading to an increase in the efficiency of transcription and translation and thus the protein yield. To test this idea, we introduced DNA nanosphere as a crowding agent in a coupled cell-free protein expression system and analyzed the subsequent protein yields. Although further study needs to be done, the results show that DNA nanosphere may in fact enhance the protein yields from cell-free expression. To functionalize DNA nanospheres as dye tracer, we attempted to fabricate dye-encapsulated DNA nanospheres with high structural stability and fluorescence signal. The effects of several engineering conditions such as photoreaction conditions and DNA building block structure were studied in order to find the optimized conditions for fabricating dye-encapsulated DNA nanospheres. It has been found that UV intensity, the presence of poly(ethylene glycol), photoinitiator concentration, and number of dyes on DNA building block monomer play a role in the structural stability of DNA and fluorescence intensity.