The inherent ecological environment of mountainous regions is highly fragile, and the degree of sustainable development is low. There has not yet been a multi-phase ecological vulnerability evaluation (EVE) study based on remote sensing (RS) and GIS for mountainous provinces, for which there is an urgent need to establish a system that is appropriate, practicable and easily operated and applied. In this study, an integrated “RS and GIS + multi-phase land use/cover change (LUCC) + practically quantitative theory and methods of EVE” approach was adopted for analysis based on the interpretation results of five phases of the land use/land cover (LULC) RS images of Yunnan, with 129 counties being considered as the evaluation units. The organic combination of quantitative multi-index comprehensive evaluation (QMCE) and qualitative comprehensive analysis (QCA) methods was adopted to perform quantitative calculations of a system of county-level evaluation indicators which includes “innate” natural ecological vulnerability (INEV), land use ecological vulnerability (LUEV) and land cover ecological vulnerability (LCEV); the degree of ecological vulnerability (DEV) was assessed for the 129 counties within the province during the five study phases (1980, 1990, 2000, 2010 and 2020). The spatiotemporal variation characteristics and laws of DEV from 1980 to 2020 in the whole province and 129 counties were revealed, aiming to provide a basis for meeting the SDGs for mountainous provinces. The results are as follows: (1) Overall, INEV is high because of the high mountains and steep slopes, and the entire province is classified as “highly vulnerable” on average. In terms of counties, more than 79.07% are classified as “moderately vulnerable”, “highly vulnerable” and “very highly vulnerable”. (2) The degree of LUEV and LCEV caused by acquired human socioeconomic activities was higher in 1980. However, after a series of ecological measures in the past 40 years, the values of DEVLU and DEVLC in the whole province and counties in 2020 have decreased to different degrees. Accordingly, the degree of overall ecological vulnerability of Yunnan province and counties decreased significantly from 1980 to 2020. The basic law of change is that the number of counties with high DEV decreases significantly, while the number of counties with low DEV increases significantly. (3) The regional difference in the DEV of Yunnan province is large. In general, the degree of ecological vulnerability is lower in the southern, southwestern, western and central areas of Yunnan and higher in the northwest high mountain canyon, northeast mountain areas and east and southeast karst areas. (4) Overall, the DEV in Yunnan province is currently still high. There is an urgent need to enhance the construction of ecological civilization across the whole province and take effective measures to protect the ecological environment according to local conditions, so as to steadily reduce the DEV.