We have recently developed densely glycosylated quantum dots (glycan-QDs) as multifunctional probes for MLGIs. The QDs are not only used as scaffolds for displaying polyvalent glycans to enhance lectin binding affinity, but also act as a donor to establish a sensitive QD-FRET (Förster resonance energy transfer) based ratiometric readout for affinity quantification. We have further harnessed the QD's nanoscale size and high contrast under electron microscopy (EM) to dissect the exact binding modes for a pair of tetrameric lectins, DC-SIGN and DC-SIGNR collectively denoted as DC-SIGN/R, hereafter. These two lectins are closely related, sharing ~80% amino acid identify, the same tetrameric architecture, and identical monovalent mannose binding motifs. Despite this, their binding properties with a polyvalent mannose-α1,2mannose capped QDs (QD-DiMan) are very different: DC-SIGN binds strongly (low to sub-nM 𝐾 𝑑 values) to QD-DiMan via simultaneous tetravalent binding, while DC-SIGNR binds much more weakly (>100-fold lower affinity) via intercross-linking. We attribute such differences to subtle orientation differences of their four carbohydraterecognition-domains (CRDs), where, all four CRDs point uprightly away from coiled-coil neck in DC-SIGN, but those in DC-SIGNR are split into two pairs and point sideways. The CRD orientation difference may also account for their distinct multivalent glycan binding and virus transmitting properties: DC-SIGN is more effective in transmitting some HIV strains than DC-SIGNR, while only DC-SIGNR, but not DC-SIGN, can effectively transmit the West Neil Virus infection. The close structural similarity and monovalent mannose specificity, yet distinct multivalent binding properties with QD-DiMan, makes DC-SIGN/R a perfect pair of model lectins to probe how binding modes determine binding thermodynamics and kinetics of MLGIs. Moreover, DC-SIGN/R play a key role in facilitating the infection of a wide range of viruses, e.g. HIV, HCV, Ebola, Zika and more recently SARS-CoV-2. Thus their MLGI biophysical parameters are of great importance and biological significance. Compared to other readout strategies, QD-FRET has the advantages of high sensitivity, simple, separation free detection in solution, and high assay robustness. In fact, QD-FRET has been widely employed in biosensing, bioanalytical and diagnostic assays, as well as bioimaging applications. Here, we have extended its use to quantify the binding thermodynamics between DC-SIGN/R and QD-DiMan by measuring their temperaturedependent affinities followed by Van't Hoff analysis. We have also studied their binding kinetics via stopped flow fluorescence, revealing that DC-SIGN binding occurs in a single step, whereas DC-SIGNR binding yields a more complex, two stage process. We have further identified that a 16 amino acid segment located at the C-terminus of DC-SIGN, which is absent in DC-SIGNR and has been shown to play an important role in DC-SIGN's ability in HIV transmission, plays a key role in defining DC-SIGN's binding thermodynamic profiles. Overall, this work has significantly extended the capacity of our glycan-QD probe, making it a powerful new platform for studying a range of biophysical parameters, mechanisms, and protein structure-function relationships for MLGIs.