Three aggregation-induced emission active fluorescent compounds, TPA-Pyr-Octane, TPA-Pyr-Br, and TPA-Pyr-Thiourea (TPA = triphenylamine pyridinium), are synthesized; their tiny differences in chemical structures result in a huge difference in cell-imaging applications. Especially, incorporating thiourea into fluorescent probes is found as a reliable strategy for mitochondrion-targeted imaging and superoxide anion tracking in living cells, which is possibly due to the presence of hydrogen bonding between thiourea and mitochondrion proteins. This finding is very useful for the design of biosensors and delivery carriers in disease treatment.