Water-related problems have become a tough issue worldwide. As a massive country with extremely large population and limited water resources, China is faced with a serious water condition, making protection of aquatic ecosystem an urgent task. Originated in China, aquatic ecological red-line is an innovation for water resources management and ecological protection. However, the study of aquatic ecological red-line is still at the exploratory stage, i.e., there is barely any research about the definition, framework and delimitation of aquatic ecological red-line. Based on the characteristics and influencing factors of aquatic ecosystem, this study explains the connotation and definition of aquatic ecological red-line. To cover major aspects of aquatic ecosystem, we propose a classification of aquatic ecological red-line which contains magnitude red-line, spatial red-line and water quality red-line, and analyze the interaction among these red-lines. Our study shows that magnitude red-line plays the leading role, whereas nearly equal attention should be paid to other two kinds of red-lines. And there is no strong relationship between spatial red-line and water quality red-line. 22 indicators are designed to transform ecological protection targets into executable constraints for riverine ecosystem. According to their importance and difficulty level to realize, these indicators are ranked and graded as primary indicators, medium indicators and advanced indicators, which provides a convenient way to find suitable positions for river protection. Taking the natural and social attributes into consideration, a delimitating method, which consists of targets determination, methods screening, index test and feasibility test, is established. Three transects in the main stream of the Huai River are chosen as examples to delimitate aquatic ecological red-lines. Considering the serious water problems in this basin, only basic targets are set for aquatic ecological red-lines. Nevertheless, the result of feasibility test reveals that failures still exist even for primary ecological demands.