Colorectal cancer (CRC) is the most common malignancy-related cause of death worldwide. The prognosis of CRC patients is strongly correlated with elevated plasma copper. The quantitative detection of plasma copper is highly essential for the field of biomedicine, and our hypothesis was that plasma copper may be a therapeutic target for treating CRC. Here, we developed metformin-based carbon dots (MFCDs). MFCDs have strong photoluminescence at 460 nm, which is quenched by Cu²⁺. Cu²⁺ concentration and the fluorescence emission intensity of MFCDs are significantly associated (linear range of 0.5–40 μM with a detection limit of 0.30 μM). Additionally, administration of MFCDs significantly inhibited the growth of CRC xenografts in mice caused by the existence of biguanide functional groups. MFCDs treatment led to the increase of reactive oxygen species and subsequent DNA damage and cell cycle arrest. This study showed that the biguanide functional groups of MFCDs chelate with Cu²⁺, resulting in the reduction of Cu/Zn-superoxide dismutase and glutathione, which eventually reduces antioxidant capacity and ultimately induces cell apoptosis. These results indicate that MFCDs provide a novel approach for anticancer therapy.