China is now facing multiple and interwoven water problems such as flooding, drought, water pollution, aquatic habitat loss and increasingly alienated human-water relationship. The solution to these problems should go beyond the single-minded engineering approach and be sought from bringing integrative hydro-ecosystem services through systematic restoration of its health and protection of its integrity. In such context, defining a relatively complete hydro-ecosystem boundary, as a bottom line for safeguarding its security and health, would lay an essential foundation for solving water issues ecologically, and also has strategic implication for current nation-wide buildup of Sponge City and Sponge National Landscape in the future. This article for the first time raises the concept of "Hydro-ecological Space Red Line",The article also proposed the red line defining methodology that is derived from hydro-ecological security pattern, specifies its technical procedure of "Hydro-ecological problem analysis-hydro-ecological process modelling and assessment-hydro-ecological security level classification-hydro-ecological security pattern identification-hydro-ecological red line drawing", and develops four red line types (for securing water supply, sustaining hydrological regulation function, supporting aquatic wildlife and conserving water cultural services, respectively). An integrative hydro-ecological red line was then formed, represented as a spatial pattern composed of landscape elements including patches, lines and areas as well as their spatial relationships that are critical in supporting hydro-ecological process. Taking Yanqi Lake as an example, an empirical study was carried out.