The natural mineral orpiment (As2S3) has long been used in traditional medicines for various diseases, although it is poorly soluble and has resulting low bioavailability. In this study, orpiment quantum dots (QDs) belonging to rare V–VI binary QDs were first synthesised through top-down and bottom-up routes, in which a mixture of ethanolamine and triethanolamine was used as a coordinating solvent. The as-synthesised orpiment QDs have a narrow size distribution, superior solubility, strong blue photoluminescence emission, and good stability. Preliminary in vitro cytotoxicity studies show that orpiment QDs are less cytotoxic for human normal dermal fibroblast cells but more potent against murine melanoma B16 cells through induction of apoptosis. Moreover, self-assemblies of orpiment QDs were fabricated through destroying the protective surface ligand layer surrounding the inner orpiment cores by addition of an acid. The underlying driving force is probably competitive reactions between the surface amine ligand and the introduced acid, leading to the exposure of the bare inner orpiment cores with high surface energy.