Reactive oxygen species (ROS) not only are byproducts of aerobic respiration, but also play vital roles in metabolism regulation and signal transductions. It is important to understand the functions of ROS in biological systems. In addition, scientists have made use of ROS to kill bacteria and tumors through a process known as photodynamic therapy (PDT). This paper provides a concise review of current molecular tools that can generate ROS in biological systems via either nongenetic or genetically encoded way. Challenges and perspectives are further discussed with the hope of broadening the applications of ROS generators in research and clinical settings.