Alkaline phosphatase (ALP) activity is regarded as an important biomarker in medical diagnosis. A ratiometric fluorescent probe is developed based on a phosphorylated chalcone derivative for ALP activity assay and visualization in living cells. The probe is soluble in water and emits greenish-yellow in aqueous buffers. In the presence of ALP, the emission of probe changes to deep red gradually with ratiometric fluorescent response due to formation and aggregation of enzymatic product, whose fluorescence involves both excited-state intramolecular proton transfer and aggregation-induced emission processes. The linear ratiometric fluorescent response enables in vitro quantification of ALP activity in a range of 0-150 mU/mL with a detection limit of 0.15 mU/mL. The probe also shows excellent biocompatibility, which enables it to apply in ALP mapping in living cells.