It's of great significance to scientifically delineate the ecological conservation redline (ECR) in karst rocky desertification areas for maintaining regionally ecological security, promoting sustainable economic and social development. Three kinds of ecosystem services such as water retention, soil conservation, and biodiversity conservation, as well as two kinds of ecological environment sensitivity indicators (soil loss and rocky desertification) were selected to construct an ECR delineation method for karst rocky desertification areas. Taking the karst rocky desertification area in Weining County of Guizhou Province as an example, combined with the Second National Land Survey data and related planning data, ECR was delineated. We analyzed the evaluation effect from three aspects:ecological land use, vegetation coverage and anthropogenic disturbance index (ADL). Results showed that the comprehensive ecosystem service function was extremely important, accounting for 40.57% of the research area. The comprehensive sensitivity level indicated that the ecological environment was sensitive, accounting for 67.86%. Finally, the area of optimized ECR in Weining County was determined to be 1496.13 km², accounting for 23.75%. The proportion of ecological land in the ECR is 20% higher than that of non-ecological land. In recent years, the implementation of the project of Grain for Green in Guizhou Province, the comprehensive control project of rocky desertification in the karst area of Weining County and the ecological restoration project in Caohai Nature Reserve, vegetation coverage increased significantly. The proportion of normalized difference vegetation index (NDVI) basic constant zone, slightly improved zone and obviously improved zone in the ECR reached 88.42%. It showed that the vegetation coverage is increasing in the ECR. The ADI in the ECR was much lower than the ADI outside the ECR. Xueshan Town and Mazha Town of Weining County need to control the ADI in the ECR, ensure that the property of ecological land within the ECR does not change, and strengthen ecological protection and supervision. The delineation of the ECR in Weining County can provide reference for the delineation and evaluation of the ECR in other rocky desertification areas.