Surface sensitive synchrotron X-ray scattering and spectroscopy are used to monitor and characterize the spontaneous formation of 2D Gibbs monolayers of thiolated single-stranded DNA-functionalized gold nanoparticles (ssDNA-AuNPs) at the vapor–solution interface by manipulating salt concentrations. Grazing incidence small-angle X-ray scattering and X-ray reflectivity show that the noncomplementary ssDNA-AuNPs dispersed in aqueous solution spontaneously accumulate at the vapor–liquid interface in the form of a single layer by increasing MgCl2 or CaCl2 concentrations. Furthermore, the monoparticle layer undergoes a transformation from short- to long-range (hexagonal) order above a threshold salt-concentration. Using various salts at similar ionic strength to those of MgCl2 or CaCl2 such as, NaCl or LaCl3, it is found that surface adsorbed NPs lack any order. X-ray fluorescence near total reflection of the same samples provides direct evidence of interfacial gold and more importantly a significant surface enrichment of the cations. Quantitative analysis reveals that divalent cations screen the charge of ssDNA, and that the hydrophobic hexyl-thiol group, commonly used to functionalize the ssDNA (for capping the AuNPs), is likely the driving force for the accumulation of the NPs at the interface.