Alkaline phosphatase (ALP) is one of essential biomarkers in mammalian tissue. Here we report a ratiometric probe for ALP, which is rationally designed and synthesized by employing ESIPT fluorophore N-(3-(benzo[d]thiazol-2-yl)-4-hydroxyphenyl)benzamide (BTHPB). The enzymatic dephosphorylation converts the probe to BTHPB, which exhibits a large spectral red-shift (120 nm), allowing extremely high sensitivity of ALP sensing at 0.004 mU/mL. The probe also shows excellent biocompatibility and has been applied for monitoring the endogenic ALP in living cells.