Surface functionalization of nanoparticles is an important determinant of their interactions with biological compartments at the nano-bio interface. In this paper, a series of multidentate zwitterionic polymeric ligands were synthesized and used to functionalize the surface of quantum dots (QDs). The structure of polymer ligands was designed by changing the molar ratio of reactants and precursors used in the reaction. A three-component micro-emulsion method was developed to improve the efficiency of ligand exchange and avoid cross-linking reactions. Highly stable, compact and biocompatible zwitterionic QDs with different surface charge densities were obtained after ligand exchange. Variation of the surface charge density of QDs was verified by zeta potential measurements. The interaction of zwitterionic QDs with different cancer and normal cell lines (KB 3-1, COLO 205 and HEK 293) was surface charge density dependent. From cell viability studies, it was shown that higher surface charge density resulted in lower cytotoxicity of zwitterionic QDs when incubated with both cancer and normal cell lines. Furthermore, the feasibility of conjugating functionalized QDs (coated with amine zwitterionic polymer ligands) with a biomolecule was demonstrated. This was exemplified by the conjugation of amine zwitterionic QDs with a cRGD peptide, which showed improved interaction of cRGD-QDs with ανβ3 integrin receptors expressed on U87MG glioblastoma tumor cells. Engineering the surface charge density and functionalization of nanoparticles, by multidentate zwitterionic ligands, provides a strategy to tune the surface properties of QDs, which impacts their cytotoxicity and cellular interaction at the nano-bio interface.