Ultraviolet-B (280−320 nm wavelength; UV-B) is one of the important reactive oxygen species (ROS) production factors in plants. The use of chlorofluorocarbons (CFCs) as refrigerants or propellants caused a serious depletion of ozone in the 1980s, which led to an increase of UV-B. Although the emissions of CFCs have decreased significantly in recent years, the ozone layer will not recover soon. The slow recovery of the ozone causes the slow decrease of UV-B intensity, and the intensity of UV-B varies with time and weather throughout the day. In the present study, typical intertidal macroalgae Ulva prolifera was exposed to moderate and extreme UV-B radiation (1 W m-2 and 5 W m-2), and the production and roles of ROS in U. prolifera under UV-B are discussed. The results are as follows: 1) ROS were mainly produced in the chloroplast under moderate UV-B radiation (1 W m-2). ROS played the signaling molecule function and led the secondary signaling pathways during this process. 2) Excessive ROS were produced in both the chloroplast and peroxisome. OH caused irreversible damage to the thalli exposed to 5 W m−2 radiation. Overall, these novel results support the idea of dual roles of ROS in U. prolifera under UV-B radiation (signaling molecules and oxidative damage). Moreover, they further highlight the role of photosynthesis in the production of ROS under UV-B.