Hydrogen peroxide (H2O2) is emerging as a new second messenger, which plays vital roles in intracellular signaling, thereby triggering physiological variations in terms of proliferation, differentiation, and migration. As known, cell mitosis has close association to the intracellular level of H2O2, which contribute to the significant effects on brain development, especially during the critical period of immaturity. Unfortunately, imaging H2O2 in a mammalian brain is still challenging. Herein, to further investigate the biological roles of endogenous H2O2 in cell mitosis, we develop a near-infrared ratiometric fluorescent probe Cy-PFS for specifically imaging endogenous H2O2 in cells and in vivo. Employing the probe Cy-PFS, we examine the critical effects of endogenous H2O2 on proliferation of cells in live hippocampal neurons cells, and our results provide strong evidence for H2O2 signaling in cell mitosis through growth factor signaling. Furthermore, we successfully demonstrate the close association of endogenous H2O2 level changes with the brain development at various stages. We envision that this present probe has potential as a promising useful chemical imaging tool for exploring the roles of H2O2 in cell mitosis.