Reactive oxygen species (ROS) often have significant roles in mediating redox modifications and other essential physiological processes, such as biological process regulation and signal transduction. Considering that H2O2 is a substantial member of ROS, detection and quantitation of H2O2 undertakes important but urgent responsibility. In this report, a bioluminescent probe for detecting H2O2 was well designed, synthesized, and evaluated. This probe was designed into three parts: a H2O2-sensitive aryl boronic acid, a bioluminescent aminoluciferin moiety, and a self-immolative linker. After extensive evaluation, this probe can selectively and sensitively react with H2O2 to release aminoluciferin. It should be pointed out that this probe is a potential bioluminescent sensor for H2O2 since it can provide a promising toolkit for real-time detection of the H2O2 level in vitro, in cellulo, and in vivo.