N,N,N',N'-Tetramethylethylenediamine (TMEDA) and ethylenediamine (EDA) were investigated in-depth in the ligand exchanges for water-soluble CdSe quantum dots (QDs). TMEDA could assist the phase transfer of QDs from apolar solvents to the aqueous solutions as stabilized by mercaptopropionic acid (MPA). We successfully maintained the stability of a series of MPA-capped QDs of different ligand densities for NMR characterizations in aqueous solutions. The proton NMR spectroscopies of MPA of the binding state were used to analyze the ligand densities on the surface of QDs, which were not explored in the past. The binding thermodynamics of the surface ligands of QDs, as analyzed using the Hill equation, demonstrated a positive promoting effect and possible interactions between ligands. EDA in the purification process underwent a spontaneous adsorption with two-stage thermodynamic behaviors as characterized by isothermal titration calorimetry. Due to the positive role of the already adsorbed ligands, excess EDA would further attach to the surface of QDs in the form of non-bonded physisorption, greatly improving the quantum yield (QY) of QDs, and the ligand of this part would almost not change the stability of QDs. We proposed a strategy for the preparation of aqueous QDs with a high QY, followed by fluorescence quenching-enhancement cycles caused by purification-adsorption operations. The strategy made it possible for the preparation of functional QDs with small molecules after purification operations. Kinetics of the sorption of ligands on the surface of QDs were determined by fluorescence spectroscopy. Modified pseudo-second-order kinetics after consideration of the ligand-ligand interaction effect could well analyze the kinetic data. This kinetic model had advantages over the previous ligand exchange model in terms of accuracy, reproducibility, and physical significance. Finally, we used the above strategy for the design of fluorescent QDs for bioimaging of lysosomes, mitochondria, and cancer cells. This work can simplify the preparation of multifunctional fluorescent QDs and avoid complicated ligand design.