Abstract: The Dongjiang River Basin has been one of the most important parts of the Pearl River Delta in Guangdong Province, southern China. But the ecological environment of the river basin has been regional fragile in recent years, due to the frequent soil erosion. It is a high demand to identify the critical ecological spaces for environmental security in the sustainable development of the main ecosystem. In this study, an evaluation system was constructed to quantitatively reveal the distribution characteristics of the critical ecological space using importance and sensitivity. Five aspects were selected to identify the regional critical ecological spaces, including the water conservation function, soil and water conservation function, biodiversity function, soil erosion sensitivity, and geological hazard sensitivity. Specifically, an evaluation model was established to fully meet the typical red soil in the ecologically vulnerable areas. The daily rainfall model in the southern mountainous area was selected to obtain the rainfall erosivity factor (R). The soil and water conservation measures factor (P) was determined to acquire various evaluation factors under the similar geographical characteristics. A local evaluation index system was also constructed to accurately identify the critical ecological space using the net primary production quantitative evaluation. A comparative analysis was carried out to verify the accuracy of the identification using Google Earth images and the territorial spatial planning data of the cities and counties in the study area. Finally, the distribution of various critical ecological spaces was explored for the ecological security of land use. The results showed that: 1) The critical ecological space was mainly distributed in the middle and lower reaches, the area of which was 16 734.48 km2, accounting for 50.49% of the total area of the basin. Among them, the urgent ecological space was mainly distributed in various nature reserves and scenic spots, the area of which was 5 481.21 km2, accounting for 16.54% of the total area of the basin; whereas, the crisis ecological space was mainly distributed in the downstream developed cities, the area of which was 11 253.27 km2, accounting for 33.95% of the total area of the basin. The critical ecological space was closer to the area of human activities than the urgent ecological space, and the urgent ecological space was close to the ecological conservation redline space in national land space planning. 2) The conflicts between land use and ecological security showed the most construction and cultivated lands were mainly distributed in the crisis ecological space, including the critical ecological space, accounting for 47.04% and 66.66% of the area, respectively. However, the construction and cultivated land were occupied only 2.48% and 17.35% of the urgent ecological space, accounting for 11.41% and 26.28% of the crisis ecological space, respectively. Each ecological space was characterized by woodland and cultivated land. The feasibility recommendations were also proposed to combine the identification and land use status for the ecological protection strategies in the Dongjiang River Basin.