With the increasing demand and scope expansion of human activities, construction and development, agriculture and forestry planting and other behaviors continue to encroach on and divide the original land system, resulting in the miniaturization and fragmentation of regional landscape patches, and ecological problems. Yunnan is not only an ecological security barrier in southwestern China but also an area with a fragile ecological environment. Study of landscape ecological security and its influencing factors in Yunnan Province is of great significance to regional, national, and even international ecological security. Taking Yunnan Province as the research object, this paper develops a landscape ecological security evaluation method in patch scale to evaluate the landscape ecological security of Yunnan Province in the past 30 years from 1990 to 2018 and uses the spatial autocorrelation method to analyze its spatio-temporal evolution law. And the main driving factors affecting landscape ecological security are identified by geographic detectors. The results show that:(1) compared with the traditionally ecological security evaluation model, the patch-scale landscape ecological security model is more sensitive to the changes of landscape ecological security and can reflect the subtle changes of landscape ecological security in the region for many years. (2) The overall ecological security cost of Yunnan Province fluctuated and increased from 1990 to 2018, in which the landscape ecological security in the area of "three screens and two belts" improved obviously, while the periphery of the urban agglomeration in central Yunnan and some areas in the karst zone in southeastern Yunnan decreased. The average Global Moran's I index of landscape ecological security in Yunnan Province is-0.293. The patches in the region are divided and the spatial distribution is discrete. (3) At present, the situation of landscape ecological security in Yunnan Province is mainly affected by population density, annual average temperature, and altitude; from the aspect of the interactive drive, the spatial distribution of landscape ecological security in Yunnan Province is the result of the joint action of natural and human factors, among which the interactive driving effect of natural factors is more obvious. The results can provide theoretical support and a scientific basis for the optimization of landscape ecological security patterns in Yunnan Province.