Ancient trade routes significantly influenced political structures, cultural exchanges, economic development, and landscape transformations across diverse regions. The Ancient Tea Horse Road, a prominent historical commercial pathway in China, is crucial for ecosystem and cultural heritage conservations. This study uses content and comparative analysis, Geographic Information System (GIS) technology, and spatial analysis to examine the spatiotemporal relationships between 13,026 settlement points identified via Google Earth and 445 recorded settlement names within the Dali Bai Autonomous Prefecture, Yunnan Province. The findings indicate that the Ancient Tea Horse Road evolved from a simple linear form to a complex radial network, reflecting its growth from local to broad regional connectivity. Settlements related to administrative, postal, and commercial functions emerged in its vicinity, underscoring the route’s impact on social structures and economic activities. The development of settlements, mainly within a 10 km radius of the route, was closely linked to its expansion, alteration, and decline, offering insights into contemporary settlement distribution patterns. This research not only enhances the understanding of the Ancient Tea Horse Road and its surrounding settlements but also supports the protection of ancient trade routes and their cultural heritage. It contributes to sustainable development by integrating ecological preservation with the safeguarding of ancient trade routes and their cultural legacies.