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Superoxide anion (O2•−) is an important reactive oxygen species (ROS), and plays critical roles in biological systems. ER stress has close relation with many metabolic diseases, and could lead to the abnormal production of ROS including O2•−. Herein, we present an ER-targeting probe (ER-Tf) for the detection of O2•− in living cells. The probe ER-Tf used triflate as the response site for O2•−, and employed p-methylbenzenesulfonamide as ER-targeting moiety. In response to O2•−, the triflate of the probe ER-Tf converted to hydroxyl group, providing strong blue emission under the excitation of ultraviolet light. The probe ER-Tf exhibited high sensitivity and selectivity to O2•−. Bioimaging experiments showed that the probe ER-Tf can be applied to detect O2•− at ER, and also demonstrated that rotenone could increase the generation of O2•− in living cells, while the O2•− level at ER showed no remarkable change during ferroptosis. [ABSTRACT FROM AUTHOR]