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Long-term labeling of the plasma membrane is crucial for visualizing membrane protein expression and morphological changes but is challenging due to the high fluidity of the plasma membrane, which can lead to probe diffusion or internalization of cells. Here, we precisely control the localization of carbon dots (M-CDs) on the plasma membrane without internalization after long-term observation under fluorescence microscopy. Adjusting the molar ratio of folic acid to o -phenylenediamine allowed fine-tuning of the water solubility and fluorescence emission of the carbon dots. Notably, carbon dots synthesized with a folic acid to o -phenylenediamine molar ratio of 1 : 10 (referred to as M-CD) exhibit excellent cell membrane targeting, likely due to the combination of suitable water-solubility and FA-FR affinity. The photostability of M-CDs is significantly superior to that of the commercial CellMask Crimson, allowing for specific recognition of folic acid receptor-positive cancer cells and minimal internalization over a period of up to 9 hours. This photostable, membrane-targeting M-CD provides a powerful tool for accurately, real-time, and non-invasively assessing the expression of folic acid receptors on cancer cell membranes and tumor metastasis., Competing Interests: The authors declare no competing interests., (This journal is © The Royal Society of Chemistry.)