Abstract In this work, based on the fact that pyrophosphate (PPi) could regulate the activity of Zn2+-dependent DNAzyme, we for the first time report a fluorescence turn-on sensing system for alkaline phosphatase (ALP) with improved sensitivity via nonprotein-enzymatic signal amplification. A catalytic and molecular beacon (CAMB) design was employed to further improve its sensitivity. Taking advantage of the strong interactions between PPi and the Zn2+, the cofactor Zn2+ was caged, and the DNAzyme activity was effectively inhibited. The introduction of ALP, however, could catalyze the hydrolysis of PPi and release free Zn2+, resulting in the activation of DNAzyme to catalyze the cleavage of the molecular beacon substrate with a remarkable increase of fluorescent signal. These optimized designs together allow a high sensitivity for ALP, with a detection limit of 20pM observed, much lower than previously reported methods. It has also been used for detection of ALP in human serum with satisfactory results, demonstrating its potential applications in clinical diagnosis.