In summary, by exploiting the versatile gold-thiol chemistry, tunable size, and powerful fluorescence quenching properties of GNPs, we have developed the glycomimetic functionalized gold nanoparticles, Gx-psDiMan, as a powerful new biophysical probe for MLGIs. We have observed that the minimal psDiMan content threshold on GNP surface to form strong MLGI with DC-SIGN in solution is dependent upon the GNP scaffold size, which can be rationalized by the CRD's footprint. We have developed a new GNP based fluorescence quenching method to quantify MLGI binding thermodynamics by combining temperature-dependent affinity measurement and Van't Hoff analysis. We have revealed that Gx-psDiMan-DC-SIGN binding is enthalpy driven, with a binding ΔH 0 of ~-95 kJ.mol -1 , ~four times that of psDiMan-DC-SIGN monovalent binding, implying that all four binding sites in each DC-SIGN are engaged in binding with Gx-psDiMan. Importantly, this binding ΔH 0 value matches well to that measured from ITC, thus verifying the credibility of our GNP based fluorescence quenching method in probing MLGI thermodynamics. We have further demonstrated that Gx-psDiMan can potently block cell surface DC-SIGN augmented EBOV-GP driven virus cellular entry with sub-nM to mid-pM level of EC50 values. Such low EC50 values place them among the most potent glycoconjugate inhibitors against DC-SIGN mediated virus entry into host cells. Moreover, we have observed that GNP scaffold size is critical towards the antiviral properties of glycan-nanoparticles. The smaller G5-psDiMan shows negative inhibition cooperativity (n = ~0.5), but the larger G13-psDiMan exhibits positive inhibition cooperativity (n > 1). As a result, the latter has achieved complete inhibition at a lower concentration than the former, despite of a higher EC50 value (i.e., 0.18 ± 0.04 vs. 0.06 ± 0.03 nM). This result highlights the critical role of inhibition coefficient "n" in determining the efficiency and viability of glycoconjugate based antiviral entry inhibitors.