Sulfur quantum dots（SQDs）， as an emerging class of non-metallic quantum dots（QDs）， have attracted much attention in the biomedical field due to their characteristics of unique biocompatibility and low cytotoxicity.However， there is still a lack of systematic studies on the interaction mechanism of their chiral surface properties with biological systems.This article successfully constructed enantiomeric SQDs（L-/D-Cys-SQDs） with mirror symmetry structure based on the chiral surface engineering strategy using L-cysteine（L-Cys） and D-cysteine（D-Cys） as chiral ligands.The results of circular dichroism（CD） analysis showed that the enantiomeric structures of L-Cys-SQDs and D-Cys-SQDs were identical.Cys-SQDs had positive and negative mirror symmetry signals at 200 nm， clarifying their differences in chiral conformation.Human breast cancer MCF-7 cells were used as a model to systematically evaluate the conformational relationship of SQDs.The results of MTT assay showed that the cell viability of the L-Cys-SQDs group after 48 h treatment with 4.0 mg/mL SQDs， was 80.4%， while that of the D-Cys-SQDs group was significantly reduced to 66.0%， indicating that the D-configuration SQDs enhance the toxicity.The results of flow quantitative analysis showed that the cellular uptake of D-Cys-SQDs was 12.1% lower than that of L-Cys-SQDs， indicating a negative correlation between the cell internalization efficiency and toxicity.The results of the DCFH-DA probe assay showed that the level of intracellular reactive oxygen species（ROS） was elevated in the cells induced by D-Cys-SQDs compared with that of L-Cys-SQDs.The “configuration-dependent toxicity” phenomenon of chiral SQDs was revealed and the phenomenon of low uptake-high bioeffects was found， breaking through the traditional cognitive framework of “positive uptake-toxicity correlation” of nanomaterials.The chiral surface engineering strategy established will provide a new idea to precisely regulate the biological effects of SQDs， with both expanding the potential of chiral nanomaterials in the diagnosis and treatment of diseases and laying the foundation for designing the functionalized QDs with low-toxicity.