Chiral nanomaterials have great potential for enhancing clinical therapeutic efficacy, attributed to the distinctive chiral selectivity in biosystems. Herein, we designed chiral iron single-atom nanoenzymes (L/D-Cys@SAFe/NC) that exhibit remarkable tumor microenvironment-triggered catalytic therapeutic activity, by simultaneously depleting GSH and generating potent reactive oxygen species in cancer cells. Under laser irradiation, they displays photothermal-enhanced catalytic activities. The study of L/D-Cys@SAFe/NC on cancer cells shows that D-Cys@SAFe/NC exhibits stronger cytotoxicity than L-Cys@SAFe/NC due to the stronger internalization ability, also supported by the higher efficiency of cancer therapy of D-Cys@SAFe/NC in vivo. This research demonstrates that the chirality of the surface ligand of the nanomaterials could exert a substantial impact on their efficacy in treating cancer, which opens a novel way for the advancement of anticancer nanomedicine.