Ecological conservation redline (eco-redline), the region with special and important ecological functions in the ecological space, must be strictly protected. According to the theory of ecology, only in a well-structured and healthy ecosystem, can the ecological function be brought into full play and the ecological security be guaranteed. Therefore, the ecosystem health of eco-redline is the basis of maintaining regional ecological security. Based on the data of land use/cover and remote sensing images in years of 2000, 2005, 2010 and 2015, this paper evaluated the ecosystem health of the eco-redline in Nanjing from five aspects: vigor, organization, resilience, ecosystem service function and anthropogenic stress. The results show that: (1) The areas of forest land, farmland, water bodies, construction land, wetland and grassland in eco-redline in the study area were 511.74 km2, 456.96 km2, 432.58 km2, 118.0 km2, 32.79 km2 and 1.56 km2, respectively. The area of forest land continued to increase, while that of farmland decreased gradually. (2) From 2000 to 2015, the average ecosystem health index of eco-redline in Nanjing was 53.83, above the sub-health level, and showing a slow growth trend. (3) The ecosystem health index of terrestrial ecosystem was higher, especially in the areas with large forest vegetation coverage, compared with aquatic ecosystem.