Discretely functionalized gold nanoparticles (AuNPs) with an amine surface functional group on each AuNP have been synthesized as a crude mixture with a significant amount of nonfunctional AuNPs. By employing the mechanoresponsive nature and reversible self-assembling properties of the mono-functionalized AuNP on a crown ether-coated gold surface, mono-functionalized AuNPs have been successfully isolated from a crude mixture. A real-time surface plasmon resonance (SPR) with organic solvent-compatible microfluidic device/instrumentation has been built together with a three-step denoising procedure for the detection of SPR absorption dip shifts with high sensitivity. With this instrument, pH-driven self-assembly and disassembly processes of the mono-functionalized gold nanoparticles (AuNPs), non-functional/mono-functionalized (crude) AuNPs, and randomly functionalized AuNPs on a crown ether-coated gold surface have been monitored simultaneously. From the real-time SPR traces, in general, the self-assembled AuNPs are stable on the surface with a solvent flow for a long period of time. These AuNPs possess different self-assembling and disassembling properties. In particular, SPR response traces obtained from mono-functionalized AuNPs reveal complete pH-driven self-assembly and disassembly processes on the gold surface. This device has enabled real-time monitoring of mono-functionalized AuNPs characteristics with the exclusion of non-functional AuNPs in a crude mixture. The SPR trace of self-assembled, randomly functionalized AuNPs indicates that only partial AuNPs were disassembled from the surface and eluted away after base treatment. Residue randomly functionalized AuNPs that were intact with the gold surface might possess higher number of functional group on each NP, rendering multivalent binding with enhanced stability towards the base. This observation has also been confirmed by atomic force microscopy (AFM). SPR monitoring of discretely functionalized AuNPs enables a real-time characterization other than the conventional transmission electron microscopy (TEM) and UV/visible absorption spectroscopic methods. The reported mono-functionalized AuNPs may find applications in single molecular detection and analysis of molecular binding events with a quantification capability with higher sensitivity.