Healthy ecosystems are the basis of social and economic development. It is of great significance to conduct ecological security assessments in rapidly urbanization areas. Based on the driving forces, pressure, state, impact, and response (DPSIR) model, five years (1995, 2000, 2005, 2010 and 2015) of remote sensing images, social and economic statistics, and field survey data were used to establish an ecological security assessment index system. The ecological security assessment of central Yunnan Province (CYP) urban agglomeration was conducted at the 1 km × 1 km pixel scale and at the county scale based on the multilevel weighted comprehensive index method. The results showed that: (1) With 2005 as the turning point, the ecological security situation in CYP first decreased and then increased. (2) The ecological security at the county scale was mainly categorized as unsafe. At the pixel scale, ecologically unsafe and relatively unsafe areas were mainly distributed in central, northern, and western CYP. (3) The ecological security deterioration and strengthened spatial distribution differences were caused by habitat fragmentation, different physical geographical conditions, and population agglomeration. These results can provide a basis for the coordination and sustainability of economic development and environmental protection in urban agglomerations with rapid urbanization.