Quantum dots (QDs) can serve as an attractive Forster resonance energy transfer (FRET) donor for DNA assay due to their excellent optical properties. However, the specificity and sensitivity of QD-based FRET analysis are prominently reduced by nonspecific DNA adsorption and poor colloidal stability during DNA hybridization, which hinders the practical applications of QDs as a biosensing platform. Here, we report subnanomolar FRET assay of DNA through the stabilization of DNA/QD interface using DNA-functionalized QDs with phosphorothioated single-stranded DNA (pt-ssDNA) as a multivalent ligand in an aqueous solution. In situ DNA functionalization was achieved during the aqueous synthesis of CdTe/CdS QDs, resulting in the maximum photoluminescence quantum yields of 76.9% at an emission wavelength of 732 nm. Conventional monothiolated ssDNA-capped QDs exhibited particle aggregation and photoluminescence (PL) quenching during DNA hybridization at 70 degrees C due to the dissociation of surface ligands. Such colloidal instability induced the nonspecific adsorption of DNA, resulting in false-positive signal and decreased sensitivity with the limit of detection (LOD) of 16.1 nM. In contrast, the pt-ssDNA-functionalized QDs maintained their colloidal stability and PL properties at elevated temperatures. The LOD of the pt-ssDNA-functionalized QDs was >30 times lower (0.47 nM) while maintaining the high specificity to a target sequence because the strong multivalent binding of pt-ssDNA to the surface of QDs prevents the detachment of pt-ssDNA and nonspecific adsorption of DNA. The study suggests that the ligand design to stabilize the surface of QDs in an aqueous milieu is critically important for the high performance of QDs for specific DNA assay.