Abstract: Improving agricultural eco-efficiency is of great significance to promote the high-quality development of Yunnan Plateau Characteristic Agriculture. Taking 16 prefectures in Yunnan Province as research areas and the period from 2008 to 2022, an index system for evaluating agricultural eco-efficiency and analyzing influencing factors with plateau characteristics was constructed. Based on the SBM model, Malmquist index, Tobit regression model, and Markov chain, the spatio-temporal characteristics, influencing factors, and future trends of agricultural eco-efficiency were examined. The results showed that: (1) From 2008 to 2022, the improvement in pure technical efficiency drove the overall increase in agricultural eco-efficiency at the provincial level, showing a spatial pattern of “higher in the northwest and lower in the southeast.” (2) Technological progress supported the overall improvement in total factor productivity, exhibiting an “NV” trend, with the highest level in Nujiang and the lowest in Qujing. (3) Urbanization and farmers income growth drove the improvement of eco-efficiency, while lower mechanization, education levels, and industrialization levels constrained it. The temporal and spatial mismatch between precipitation and crop needs affected the stability of eco-efficiency. High fertilizer application intensity emerged as one of the key factors restricting agricultural eco-efficiency. (4) Eco-efficiency showed a pronounced “spatial low-efficiency lock-in” effect and high volatility. In the long term, there is a clear tendency for agricultural eco-efficiency to converge toward medium to high levels.