Considering that hydrogen peroxide (H2O2) plays significant roles in oxidative stress, the cellular signal transduction and essential biological process regulation, the detection and imaging of H2O2 in living systems undertakes critical responsibility. Herein, we have developed a novel two-photon fluorescence turn on probe, named as Pyp-B for mitochondria H2O2 detection in living systems. Selectivity studies show that probe Pyp-B exhibit highly sensitive response toward H2O2 than other reactive oxygen species(ROS) and reactive nitrogen species (RNS) as well as biologically relevant species. The fluorescence colocalization studies demonstrate that the probe can localize in the mitochondria solely. Furthermore,as a bio-compatibility molecule, the highly selective and sensitive of fluorescence probe Pyp-B have been confirmed by its cell imaging application of H2O2 in living A549 cells and zebrafishes under the physiological conditions.