Graphical abstract The introduction of chiral enantiomers generated chiral β-HgS QDs, which showed chirality inversion compared with the corresponding chiral ligands. Their chiroptical activity, good cytocompatibility, near-infrared optical absorption, near-infrared fluorescence emission and high-performance photothermal conversion implied that these chiral β-HgS quantum dots have potential to be applied in biotechnology and bio-medicine. Abstract β-HgS quantum dots (QDs) have drawn enormous attention due to the size-tunable bandgap and the lowest quantum state in conduction band which have been applied to semiconductor transistor and photodetector. Though β-HgS is the essential component of Tibetan medicine, the potential toxicity of β-HgS limits its applications, especially in bio-application. Herein, chiral biomolecule enantiomers N -isobutyryl- L (D)-cysteine (L (D)-NIBC) and L (D)-cysteine (L (D)-Cys) were introduced into HgCl 2 and Na 2 S aqueous solution to synthesize chiral β-HgS QDs in one-pot, which significantly improved their water-solubility and cytocompatibility. Notably, all chiral β-HgS QDs showed none cytotoxicity even at high concentration (20 mg·L−1), and the cytocompatibility of D -β-HgS QDs was better than corresponding L -β-HgS QDs at the concentration of 20 mg·L−1. This cytotoxicity discrimination was associated with the chirality inversion of chiral β-HgS QDs compared with the corresponding chiral ligands. In-situ real-time circular dichroism (CD) monitoring indicated that the chirality of β-HgS QDs originated from the asymmetrical arrangement of chiral ligands on the achiral core surface. Their chiroptical activity, near-infrared optical absorption (800 nm), fluorescence emission (900–1000 nm), high-performance photothermal conversion and good cytocompatibility, implied chiral β-HgS QDs could be used as a candidate material for photothermal therapy or a near-infrared fluorescent probe in organism, which brings a novel insight for bio-application of β-HgS QDs. [ABSTRACT FROM AUTHOR]