Hydroxyl radical (·OH)， as the most deleterious reactive oxygen species， is believed to be the etiological agent for many diseases and aging. An altered ·OH level has been confirmed in certain types of superoxide dismutase (SOD) mutation， but the regulation of ·OH by SOD in situ is still controversial or unclear because of the lack of effective tools to detect ·OH in the biological environment. Herein we report the first two-photon excitable molecular probe (P2) for ·OH， which is able to track the subtle fluctuation of ·OH level both in vitro and in vivo with high sensitivity and specificity. The probe was successfully applied to visualize ·OH variations in a variety of SOD1-involved biological processes， confirming that the inhibited enzymatic activity and down-regulated expression of SOD1 both lead to elevated intracellular ·OH level. This is the first report to visually reveal the relationship between SOD1 and ·OH level with a molecular tool.