Cell apoptosis is a biochemical and molecular pathway essential for maintaining cellular homeostasis. It is an integrated process involving in a series of signal transduction cascades. Moreover, the apoptotic pathways may be initiated inside various subcellular organelles. Increasing evidence indicates that hydrogen peroxide (HO) is closely related to cell apoptosis, particularly in the mitochondria. However, during the apoptotic process, the synergetic variation of HO levels in different compartments is seldom explored, particularly in two important organelles: mitochondria and endoplasmic reticulum (ER). To solve this problem, we developed two new organelle-specific fluorescent probes termed and that can detect HO in mitochondria and ER, respectively or simultaneously. Experimental results demonstrated that and display distinguishable excitation and emission spectra, as well as excellent organelle targeting capabilities. Therefore, we used and to successfully image exogenous or endogenous hydrogen peroxide in the mitochondria and ER. Interestingly, during diverse apoptotic stimuli, dual-color fluorescence imaging results revealed that the changes of HO levels in mitochondria and ER are different. The HO levels are enhanced in both the mitochondria and ER during the l-buthionine sulfoximine (BSO)-treated cell apoptosis process. During mitochondria-oriented apoptosis induced by carbonyl cyanide -chlorophenylhydrazone (CCCP) or rotenone, HO levels prominently and continuously increase in the mitochondria, whereas the ER HO levels were found to rise subsequently after a delay. Moreover, during ER-oriented apoptosis induced by tunicamycin, ER is the major site for overproduction of HO, and delayed elevation of the HO levels was found in the mitochondria. Altogether, this dual-probe and multicolor imaging approach may offer a proven methodology for studying molecular communication events on HO-related apoptosis and also other physiological and pathological processes within different subcellular organelles.