Reactive oxygen species (ROS) can mediate cellular signal transduction, destroy hazardous foreign pathogens, regulate transcriptional activities and therefore are essential in normal cellular physiology. On the other hand, inadequate control of ROS production can induce peroxidation of essential biomolecules and result in cellular dysfunction. In the worst condition even cell death ensues. Therefore, they have great implications in the initiation and progression of numerous diseases. Chemical instability of ROS limits its direct detection and the dynamics remain poorly studied. To study the ROS dynamics, the endogenous or exogenous redox-sensitive fluorophores provide a good chance to detect the real-time changes in in vivo or in vitro settings. Here these methods are reviewed and their potential applications are discussed.