Ecological suitability evaluation is a critical component of regional sustainable development and construction, serving as a foundation for optimizing spatial patterns of regional growth. This is particularly pertinent in karst mountainous regions characterized by limited land resources and heightened ecosystem vulnerability, where a quantitative assessment of ecological suitability for land development is both crucial and urgent. Based on the fundamental principles of structural and functional dynamics in landscape ecology, this study focuses on Gui’an New Area, a designated urban development zone situated in the karst landscape of Guizhou Province. An index system was established encompassing three dimensions: ecological elements, ecological significance, and ecological resilience, utilizing the integrated ecological resistance (IER) model to evaluate the suitability of regional development and construction. The results reveal that the eastern region exhibits higher suitability compared to the central and western regions, with the northwest region demonstrating the lowest suitability overall. Relatively speaking, the evaluation of geological environment suitability and the comprehensive ecological constraints associated with development and construction indicates that the areas currently planned and ongoing reflect flat terrain and low ecological risk. Furthermore, within the scope of ecosystem dynamic adaptation, developmental activities in these regions exert minimal impact on the natural ecosystem, thereby demonstrating a high suitability for development and construction. In terms of future key development zones, areas with gentle slopes ranging from 8 to 15 degrees are recommended, aligning with the actual requirements for cultivated land protection. The total area designated as prohibited development zones constitutes the smallest proportion, representing only 9.45%, which is significantly lower than that of priority development zones (38.75%) and moderate development zones (22.45%). From the perspective of landscape ecology, this paper provides a comprehensive investigation into the ecological suitability evaluation system for development and construction in the karst regions of Southwest China, offering valuable insights for assessing ecological suitability in similar areas.