The monitoring of alkaline phosphatase (ALP) activity in different tissues is significant for disease diagnosis and therapy. However, the time-resolved in vivo sensing of ALP activity remained unresolved. Herein, a novel red-near infrared fluores-cent ALP probe (Cl2-BDCM-ALP) based on dichloro substituted dicyanomethylene-4H-chromene derivative was designed and synthesized with high fluorescence efficiency and stability under biological pH range. By using Cl2-BDCM-ALP, ALP activity under acidic microenvironment such as tumor site can be sensitively imaged, which cannot be achieved by some previously reported ALP probes. By further loading the Cl2-BDCM-ALP into a near-infrared (NIR) light-responsive nanocon-tainner, time-resolved long-term imaging of ALP activity was facilely achieved with noninvasive NIR light remote control. Time-resolved variation of ALP activity of the drug induced acute liver injury mice was successfully monitored in vivo for the first time. This strategy holds great promise in the in situ ALP detection under broad pH range with temporal resolution.