Reactive oxygen species (ROS) are a class of highly reactive oxygen-containing intermediates that are ubiquitous in lake waters, mainly including superoxide radical (O2?·), hydrogen peroxide (H2O2), hydroxyl radical (·OH), singlet oxygen (1O2), and triplet excited dissolved organic matter (3DOM*). In lake ecosystems, ROS can be generated through multiple pathways, including photochemical reactions, microbial metabolism, metal-mediated redox reactions, and sediment–water interface processes, and are jointly regulated by water depth, thermal stratification, dissolved organic matter composition, as well as inorganic ions and metal cycling. In shallow lakes, stronger light penetration and more intensive water mixing facilitate the dispersion of ROS throughout the water column and support relatively high concentration levels. In contrast, ROS in deep stratified lakes usually exhibit pronounced vertical heterogeneity, with photochemical production dominating in the surface layer, whereas interface-related processes and dark biological processes become increasingly important in the thermocline and bottom waters. ROS play important roles in lake water quality and ecosystem evolution. They participate in the cycling of biogenic elements, including carbon, nitrogen, phosphorus, iron, and manganese, influence the attenuation and transformation of pollutants, and affect lake ecosystem structure by regulating community composition and biological metabolism. Owing to their strong oxidative capacity and environmental compatibility, ROS also show broad application potential in lake pollution control and ecological restoration, including ROS-enhanced degradation and purification of water contaminants, as well as targeted ROS generation for precise regulation and restoration of lake ecosystems. It should be noted that the behavior and eco-environmental effects of ROS in lakes are closely related to their formation pathways, steady-state concentrations, and dominant species. Differences among ROS in oxidative capacity, reaction selectivity, and spatial distribution determine their distinct roles in lake waters. This review systematically summarizes the spatiotemporal occurrence characteristics, biotic and abiotic formation mechanisms, and eco-environmental effects of ROS in lake waters, and further discusses future research directions, with the aim of providing theoretical guidance and technical support for understanding lake habitat evolution, assessing ecosystem status, and improving the control and management of polluted lakes.