Hydrogen peroxide (H2O2) is one of the important reactive oxygen species (ROS), which is closely related to many pathological and physiological processes in living organisms. Excessive H2O2can lead to cancer, diabetes, cardiovascular diseases, and other diseases, so it is necessary to detect H2O2in living cells. Since this work designed a novel fluorescent probe to detect the concentration of H2O2, the H2O2reaction group arylboric acid was attached to the fluorescein 3-Acetyl-7-hydroxycoumarin as a specific recognition group for the selective detection of hydrogen peroxide. The experimental results show that the probe can effectively detect H2O2with high selectivity and measure cellular ROS levels. Therefore, this novel fluorescent probe provides a potential monitoring tool for a variety of diseases caused by H2O2excess.