Macao Special Administrative Region is one of the most densely populated regions in the world, and the human-land relationship is tense. The fast development of the city and economy resulted in the imbalanced pattern of human space and ecological space, and serious problems of urban ecology and human settlement environment. In the context of building a beautiful Bay Area and a beautiful Macao, and people’s demand for a better living environment, it is particularly important to control and maintain the existing vitally functional ecological space, and to protect and improve the highly sensitive ecological space by delimiting the urban ecological conservation redlines, so as to maintain regional ecological security. Based on the survey of the terrestrial ecosystem in Macao and by combining the characteristics of the city and natural resources, this paper constructed a framework for the delineation of urban ecological conservation redlines from four aspects, i.e., ecosystem service functions, ecosystem sensitivity, biodiversity distribution and existing ecological protection zones. We selected and evaluated five types of regulation services (including water conservation, rain and flood regulation, heat island regulation, soil conservation, and coastal protection) and leisure and recreation in ecosystem services, and analyzed distribution of the sensitivity of water and soil erosion and the coastal storm surge. The results were further combined with the distribution of habitats of the species with high conservation value in Macao and the scope of the existing protected areas, and the urban ecological conservation redlines were finally delineated. The total area within the delineated Macao's urban ecological conservation redlines was 6.71 km², accounting for 19.79% of the study area, which covered 44% of the ecological space, 12.4% of the coastline, all the important species habitats and one third of the ancient famous trees. Corresponding protection and management measures were also provided according to different kinds of land parcels in this ecological conservation redlines. The proposed frame and method of urban ecological redlines delineation could also provide reference for conservation, restoration and management of important urban ecological spaces in other cities.