Based on the zoning regulation which integrated climate, vegetation and soil as a complex, a regional eco-security index system consisting of regional physical background, ecosystem stability, landscape structure, and external interference was established, and, with the support of geographical information system technology and fuzzy mathematics, a set of regional eco-security assessment methods was constructed. By means of these methods, the eco-security index of southwest Beijing in 2004 was calculated based on the recent 30 years background data of climate and soil. The results showed that in southwest Beijing, the eco-security degree was higher in its central area and western hilly-shallow hilly areas, but lower in its southern and southeastern plain areas. The mining activity in southwest Beijing had threatened the environment, and should be stopped immediately to maintain the regional eco-security. It was proved in the case study that the assessment index system and related methods established in this paper could solve the existing problems in assessing regional eco-security, and the assessment results could reflect the regional ecological issues and the status of eco-security at different locations within the region.