A key means of promoting the high-quality development of karst areas is the maintenance of the area’s ecological security. A full recognition of the special ecological function of karst areas, as well as their significance to the surrounding region’s ecological, economic, and social development, is crucial in strengthening the overall strategic deployment of the national ecological construction and the protection and sustainable development of karst landscapes around the globe. In this study, the karst landscape of Puzhehei, Qiubei County, Wenshan Prefecture, Yunnan Province, China, was used as the research object. This study identified ecological source sites through a combination of morphological spatial pattern analysis and landscape connectivity assessment. As a result, 10 factors were selected to construct a comprehensive ecological resistance surface from the natural environment and socio-economic perspective; the resistance surface was corrected by combining the sensitivity of rocky desertification. An ecological corridor and ecological nodes were identified to construct the ecological security pattern based on the minimum cumulative resistance model and circuit theory. The results show that (1) the source areas of the Puzhehei karst landscape ecological protection comprised 11 core area patches with the landscape connectivity index of (dPC) ≥ 10, with a total area of 166.6572 km2, which constituted 46.06% of the total study area, and the ecological source area totaled 77.275 km2, or 21.36% of the total study area; (2) there were 78 potential ecological corridors in the Puzhehei karst region, with a total length of 545.186 km, including 12 key corridors and 66 general corridors; (3) a total of 51 ecological nodes were identified, including 11 “source-type ecological nodes”, 30 “ecological pinch points”, and 10 “ecological obstacles”, including 16 key ecological nodes. This study provides a theoretical basis for the integration of Puzhehei Nature Reserve, as well as a reference for the ecologically sustainable development of similar karst areas.