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Fluorescence microscopic analysis of checkpoint protein expression is capable of predicting clinical outcomes for checkpoint blockade immunotherapy. However, accurate detection of their expression levels is hindered by fluorophore photobleaching and cell autofluorescence. We now develop a sensitive and robust fluorescence microscopy method that uses antifade graphite-structured carbon dots (GCDs) on a plasmonic Ag substrate (named ACPAS) for the accurate detection of checkpoint proteins in immunotherapy. In ACPAS, a Ag substrate is used