As the chemical by-products of cell metabolism, reactive oxygen species (ROS) are widely involved in signal transduction of physiological processes. However, when redox homeostasis is out of balance, excessive production of ROS may lead to or promote multiple pathologies. Currently, the diverse chemical properties of ROS and their biological mechanism are still poorly understood. Therefore, powerful tools need to be exploited to elucidate the chemical biology of ROS. Non-invasive fluorescence imaging techniques have gradually matured for tracking of active molecules. This chapter summarizes the fluorescent sensors for ROS in living cells and in vivo.