Large-scale anthropic conversion of natural ecosystems into human-dominated countryside ecosystems has led to biodiversity loss worldwide. However, how countryside landscapes affect biodiversity is still controversial, especially in karst regions lack of relevant studies. To answer this question, we analyzed the impact of countryside landscapes on biodiversity changes in the South China Karst, featured as a World Natural Heritage site of subtropical karst landscape and rich biodiversity. We presented a new framework to examine the impact of countryside landscapes on biodiversity change at a semi centennial-scale using three indicators: biodiversity cluster characteristics, the barrier of villages to biodiversity, and the barrier of arable land to biodiversity. Unmanned aerial vehicle (UAV) images, satellite remote sensing data, and field survey data were combined during the process. Results revealed that plant biodiversity was positively correlated to vegetation restoration, and vegetation took approximately 80 years to recover to a stable state. Plant biodiversity spatial points are densely distributed at karst peaks, with an average distance of only 420 m–480 m. Small villages create no apparent barriers to biodiversity, with an average barrier distance of about 450 m–530 m. Arable land intersecting with forest patches connects biodiversity spatial points. Hence, the expansion of countryside landscapes in karst depressions was restricted by karst peaks, and patches of artificial arable land and forest connect the natural reserved forest at karst peaks, which both promote local spatial heterogeneity and sustain local biodiversity. This study enriches the application of countryside biogeography theory in karst regions and provides evidence for integrating biodiversity conservation into sustainable planning in broader areas. We strongly recommend that a heterogeneous landscape pattern similar to karst peak forests, such as the distance between biodiversity spatial points and the barrier distance to villages both around 450–500 m, would be conducive to biodiversity preservation.