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D-amino acids (DAAs) are indispensable in regulating diverse metabolic pathways. Selective and sensitive detection of DAAs is crucial for understanding the complexity of metabolic processes and managing associated diseases. However, current DAA detection strategies mainly rely on bulky instrumentation or electrochemical probes, limiting their cellular and animal applications. Here we report an enzyme-coupled nanoprobe that can detect enantiospecific DAAs through synergistic energy transfer. This nanoprobe offers near-infrared upconversi