Excessive or unfolded proteins accumulation in endoplasmic reticulum (ER) will cause ER stress, which has evolved to involve in various metabolic diseases. In particular, ER stress plays an important role in the pathogenesis of diabetes. Both ER stress and course of diabetes accompany oxidative stress and production of reactive oxygen species (ROS), among which superoxide anion (O) is the first produced ROS and has been recognized as cell signaling mediator involved in the physiological and pathological process of diabetes. Hence, the development of effective monitoring methods of O in live cells and in vivo is of great importance for ascertaining the onset and progress of related diseases. Herein, a new endoplasmic reticulum-targeted two-photon fluorescent probe termed ER-BZT is designed and synthesized for imaging of O. The probe ER-BZT shows high sensitivity, selectivity, stability, and low cytotoxicity. Based on these superior properties, the rise of O levels in endoplasmic reticulum induced with different stimuli is visualized by one- and two-photon fluorescence imaging. Most importantly, by utilizing ER-BZT, the two-photon fluorescence imaging results demonstrate that the endogenous O concentration in abdominal or hepatic tissue of diabetic mice is higher than that in normal mice. Meanwhile, after treated with metformin, a broad-spectrum antidiabetic drug, the diabetic mice exhibit depressed O level. The proposed two-photon probe, ER-BZT might serve as perfect tool to image the O fluctuations and study the relevance between O and various diseases in live cells and in vivo.