Ecological vulnerability (EV) assessment is an important means of reflecting the evolutionary process of the local ecological environment, which is crucial for ecological security. The current EV assessment still faces challenges in comprehensively revealing its spatio-temporal change characteristics due to diversity, multi-scale complexity and non-linearity of ecosystems. In this study, we comprehensively revealed the spatio-temporal change features and driver mechanisms of EV in Yunnan Province (YP) using methods such as breaks for additive seasonal and trend (BFAST) and Geodetector. According to the findings, (1) the ecological vulnerability index (EVI) in YP decreased by 0.0265 on the raster scale between 2000 and 2018, and the ecological vulnerability level (EVL) was mainly dominated by level III. At the city scale, EVL in YP was dominated by level IV. At the basin scale, the average EV of the Jinsha River basin was much greater other basins. (2) The linear trend of EV in the YP mainly showed an insignificant decrease, mostly concentrated in the YP’s eastern region. The non-linear trend mainly showed a monotonous decrease, with the mutation time concentrated in 2009. The future persistence trend of EV under different coupling modes was dominated by anti-persistence decrease (Sen-MK + Hurst) and anti-persistence monotonous decrease (BFAST + Hurst), with an area percentage of 40.19 % and 25.13 %, respectively. (3) Gross primary productivity was the priority factor influencing YP’s EV (q = 0.4137). This study not only enriches the cases of EV assessment studies in the high mountain valley area but also bridges the gap of analysing the multi-spatial scale characteristics and change trends of regional EV.