The generation of compact quantum dots (QDs) probes is of critical importance for visualizing molecular interaction occurring in biological context, particularly when using the Forster resonance energy transfer (FRET) approach. This Article reports novel water-soluble compact CdSe/ZnS QDs prepared by ligand exchange reaction using thiolated nitrilotriacetate (NTA). The resulting NTA-QDs revealed higher stability and remarkable conjugation efficiency compared to the other QDs prepared with different ligands by using the ligand exchange method. The Ni-NTA group is a well-known binding moiety for the detection and purification of oligohistidine-tagged recombinant proteins. We demonstrated that NiNTA-QDs prepared by Ni2+ complexation exhibited highly specific binding ability toward 6-histidine (His)-tagged peptides present in various experimental conditions (buffer solution, agarose beads, and HEK cells). Importantly, the compact NiNTA-QDs serve as an efficient FRET donor. These results suggest that the stable and highly selective multifunctional NTA-QDs can he useful for labeling and tracking molecular interactions within biological context.