DNA-functionalized gold nanoparticles (AuNPs) often encounter various small molecules and ions such as backfilling agents, bifunctional cross-linkers, stabilizers, and molecules from biological fluids both during and after the DNA conjugation process. Small molecules and ions can influence the stability and property of the conjugate, but such interactions are yet to be fully explored. In this work, eight important molecules were studied and compared, including tris(2-carboxyethyl)phosphine hydrochloride (TCEP), 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP), 4-maleimidobutyric acid N-hydroxysuccinimide ester (GMBS), 6-hydroxy-l-hexanethiol (MCH), L-glutathione (GSH), bromide (BC), bis(p-sulfonatophenyl)phenylphosphine (BSPP), and thiocyanate (SCN-). Depending on the size, charge, and adsorption affinity on the AuNPs, they can either stabilize or destabilize the AuNPs. Their ability to displace thiolated DNA from AuNPs follows the order of MCH > SPDP > GSH > SCN- > TCEP > Br- > BSPP > GMBS. BSPP has the best stabilization effect for the colloidal stability of AuNPs, while it does not displace the adsorbed DNA. TCEP can be adsorbed on AuNPs and enhance the adsorption of A/C rich DNA in low-salt conditions. This work indicates that the effects of small molecules and ions cannot be ignored when studying the DNA-functionalized AuNPs, which ensures optimal applications and correct interpretation of the data.