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As an important biomarker, alkaline phosphatase (ALP) is one of the most commonly assayed enzymes in clinical practice. Here a novel turn-on fluorescent nanoswitch for ALP assay was suggested. The nanoswitch was easily constructed via two high-affinity ligands between GSH and Al3+ and PPi and Al3+ based on the difference in the affinity. The primary fluorescence of as-prepared GSH capped Cu nanoclusters (NCs) turned on first upon the Al3+ addition due to the Al3+ induced aggregation induced emission (AIE) enhancement based on the high affinity of GSH and Al3+. The presence of PPi then made Al3+ desorb from the surface of GSH capped Cu NCs due to the higher affinity of PPi and Al3+. As a result, the fluorescence of the Cu NCs was quenched. ALP could hydrolyze PPi into phosphate, destroying the PPi-Al3+ complex and releasing Al3+. Thus, the Al3+ binds to GSH again and the fluorescence was restored. The nanoswitch was demonstrated to be sensitive and selective for ALP assay and was successfully used for the ALP assay in the human serum. Image 1 • A luminescence turn on Cu NCs-based nanoswitch for ALP assay was proposed. • The developed nanoswitch was based on Al3+-PPi interaction and Cu NCs with AIE properties. • The proposed nanoswitch was cheap, sensitive, selective and high stability with a LOD of 0.15 U/L. • The nanoswitch was successfully used for the ALP assay in the human serum. [ABSTRACT FROM AUTHOR]