Abnormal alkaline phosphatase (ALP) levels in the human body are closely associated with various diseases, particularly hepatobiliary diseases and bone diseases. Herein, we constructed a ratiometric sensor based on Förster resonance energy transfer (FRET) using strongly photoluminescent copper nanoclusters (Cu NCs) for the detection of ALP with high sensitivity and specificity. The cysteine-stabilized Cu NCs (Cys-Cu NCs) were synthesized through a ligand-exchange reaction and core-size etching focusing, which displayed bright photoluminescence (PL) with a quantum yield (QY) of 10.5 %. Multispectral characterization indicated that zwitterionic cysteine ligands without obvious steric hindrance could significantly enhance intra/inter-ligand-involved charge transfer, leading to a significant increase in fluorescence emission (∼14 folds) compared to precursor Cu NCs. A FRET-based ratiometric sensor for ALP detection was constructed by combining Cys-Cu NCs with a Cu