Attaching thiolated DNA to gold nanoparticles (AuNPs) is a highly important and useful reaction for many applications. Various methods such as adding salts, acids, polymers, and surfactants have been developed to facilitate the reaction. Recently, it was reported that a very high DNA density can be achieved simply by freezing AuNPs with the DNA without any other reagents. DNA oligonucleotides are also known to stretch and align upon freezing. In this work, a set of experiments were performed with a fluorophore and thiol dual-labeled DNA, and the DNA loading density and colloidal stability of AuNPs were measured. The initial salt concentration was unimportant, and even 0.1 mM Na allowed around 100 DNA attached to each 13 nm AuNPs. On the other hand, a high DNA concentration of 3 μM was needed to achieve the high DNA density and good colloidal stability of AuNPs. When the thiolated DNA was forced in stable secondary structures, the attachment was low, and preadsorbed DNA also inhibited the DNA attachment by the freezing method. Overall, nonstructured thiolated DNA strands need to align by freezing and quickly attached through the ends of the DNA. This work illustrates practical experiment design conditions and offers fundamental surface science insights for the DNA attachment by freezing.