It is challenging to design and characterize molecular recognition pair consisting of both targeting and imaging property and it is also of particular interest to inquire into the limits of the affinity that can be achieved with designed bio-conjugate. This paper presents functionalization of luminescent quantum dots (QDs) employing multivalent dendrimer molecules conjugated with biotin as targeting moiety and photo-physical and thermodynamic behavior of this bio-conjugate in terms of highly affinic binding with avidin. It is demonstrated that fluorescence intensity of QDs remained almost unaffected when synthesized conjugate interacts with avidin and this is considered to be an important requisite for applications in imaging and sensing. The observed changes in average emission lifetime as well as values of lifetime components of avidin indicate the binding of biotin with tryptophan moiety of avidin. Calorimetric investigation reveals that binding of avidin with biotin-dendrimer conjugate is exothermic process accompanied with favorable enthalpy (ΔH = −21.6 kcal/mol) and unfavorable entropy (ΔS = −43.7 cal/mol/deg). These findings may help design of multiplexed assays where dendrimer conjugated QDs based probe molecules are immobilized via the avidin-biotin interactions which can have implications in development of immuno-histochemical techniques.