Azide-oligo(ethylene glycol)-terminated self-assembled monolayers (N3-OEG-SAMs) are promising interfacial structures for surface functionalization. Its many potential applications include chemical/bio-sensing and construction of surface models owing to its cross-coupling activity that originates from the azide group and oligo(ethylene glycol) (OEG) units for non-specific adsorption resistance. However, there are only a few studies and limited information, particularly on the molecular-scale structures and local cross-coupling activities of N3-OEG-SAMs, which are vital to understanding its surface properties and interfacial molecular design. In this study, molecular-scale surface structures and cross-coupling activity of azide-hexa(ethylene glycol)-terminated SAMs (N3-EG6-SAMs) were investigated using frequency modulation atomic force microscopy (FM-AFM) in liquid. The N3-EG6-SAMs were prepared on Au(111) substrates through the self-assembly of 11-azido-hexa(ethylene glycol)-undecane-1-thiol (N3-EG6-C11-HS) molecules obtained from a liquid phase. Subnanometer-resolution surface structures were visualized in an aqueous solution using a laboratory-built FM-AFM instrument. The results show a well-ordered molecular arrangement in the N3-EG6-SAM and its clean surfaces originating from the adsorption resistance property of the terminal EG6 units. Surface functionalization by the cross-coupling reaction of copper(I)-catalyzed azide-alkyne cycloaddition was observed, indicating a structural change in the form of fluctuating structures and island-shaped structures depending on the concentration of the alkyne molecules. The FM-AFM imaging enabled to provide information on the relationship between the surface structures and cross-coupling activity. These findings provide molecular-scale information on the functionalization of the N3-EG6-SAMs, which is helpful for the interfacial molecular design based on alkanethiol SAMs in many applications.