Scientifically delineating and strictly protecting the ecological protection red line (EPRL) are essential strategic measures for the Chinese government to construct ecological civilization. It is of considerable significance to promote the sustainable development of land space. This paper puts forward four fundamental issues of the EPRL by analyzing the connotation, objectives, and delimitation criteria of the EPRL system. Aiming at the problems of biodiversity reduction, habitat patch fragmentation, and conflict between agriculture and ecological space in Middle-Lower Yangtze Plain, the regional differences of the EPRL are considered. Taking Jiangsu Province as the study area, this paper discussed the corresponding EPRL delimitation method based on the framework of ecological security pattern, considering five ecosystem service functions of habitat maintenance, climate regulation, water conservation, food supply, and soil conservation. The results show that 1) eco-source areas in Jiangsu Province mainly compose of rivers, lakes, tidal flats and woodlands, and the spatial patterns of ecosystem services are significantly different. Limited by the extensive coverage of farmland and the developed network structure of the river system, the source patches are severely fragmented. 2) Jiangsu EPRLs are mainly composed of wetland, forest, and grassland, agricultural land, etc. The core area of the EPRL is mainly wetland, while the corridors and nodes are mainly agricultural lands. The EPRL can be divided into three levels according to the landscape size of eco-source, of which grade I EPRL accounts for 72.87% and can basically represent the core EPRL patches. 3) The establishment of ecological corridors and ecological nodes helps to improve the connectivity of habitat patches. There are 47 ecological corridors in Jiangsu Province, including 33 critical ecological corridors and 14 potential ecological corridors, with total lengths of 1296.27 km and 1726.33 km, respectively. The results can provide innovative ideas for the EPRL delimitation in Middle-Lower Yangtze Plain and hope to have reference value for the system reform of land space management and control under the restriction of the EPRL system.