Metallic nanoparticles such as gold and silver are known to exhibit localized surface plasmon resonance (LSPR). Being able to form well defined nanoassemblies of metallic nanoparticles in solution phase can produce LSPR coupling and shift, which represents a unique plasmon signature and have been used for the detection of nanoassembly formation. While most of the existing works focus on nanoassembly formation on a substrate surface, here, we investigated the formation of DNA-modified gold nanoparticle (nAu-DNA) nanoassemblies in bulk solution. Subsequently the nanoassemblies were allowed to bind on a glass substrate in order to study correlations among the LSPR wavelength shift, the plasmon color change, and the nanoassembly structure. We observed that the hybridization percentage of the complementary 50 nm nAu increased with rising nAu concentration, longer hybridization time, and longer complementary duplex DNA length. In addition, due to lower scattering yield and smaller surface area from 10 nm nAu, the 50 nm/10 nm hetero-size system displayed limited observable LSPR shift compared to 50 nm/20 nm hetero-size system, which in turn was inferior to the 50 nm/50 nm homo-size system. For the hetero-size systems, reducing the surface density of ssDNA on the 20 nm and 10 nm nAu also significantly reduced the hybridization percentages. Overall, this study allows us to understand how different experimental parameters can impact the assembly of nAu-DNA probes, particularly the limitation in using smaller size nAu (10 nm) for LSPR study.