Semiconductor quantum dots (QDs) have traditionally been synthesized in organic phase and transferred to aqueous solution by functionalizing their surface with silica, polymers, short-chain thiol ligand, or phospholipid micelles. However, these complex steps result in i) a reduction of the quantum yield (QY) of QDs, ii) partial degrdation of the QDs, and iii) a drastic increase in the hydrodynamic size of QDs, which may hinder their biomedical applications. In this work, the fabrication and applications of cysteine-capped CdTe/ZnTe QDs, which are directly synthesized in aqueous media, as optical probes for specific targeting of pancreatic and esophageal cancer cells in vitro are reported, as well as their capability for in vivo imaging. The CdTe/ZnTe QDs are synthesized in a one-pot method and capped with amino acid cysteine, which contains both carboxyl and amine functional groups on their surfaces for bioconjugation. The fabricated QDs have an ultrasmall hydrodynamic diameter (3–5 nm), possess high QY (52%), and are non-toxic to cells at experimental dosages. Confocal imaging is used to demonstrate a receptor-mediated uptake of antibody-conjugated QDs into pancreatic cancer cells in vitro. In vitro cytotoxicity studies (MTS-assay) show that the IC50 value of these QDs is ≈160 µg mL−1, demonstrating low toxicity. In addition, the QDs are used for small-animal imaging where the in vivo biocompatiblity of these QDs and their clearance following systemic injection is studied.