Exploring the changes in ecosystem service value (ESV) caused by land use transition is important for regional ecological protection. According to the land use data from 2000 to 2020, the alterations in the land use and ESV in Yunnan over the past 20 years were calculated and analyzed. At the same time, spatial autocorrelation analysis was established to analyze the spatial relationships of ESV in 16 states and cities. The results show that from 2000 to 2020, cultivated land, grassland and unused land are on a decreasing trend, while forest land, water body and built-up land are on an increasing trend, with the largest change in built-up land. The total ESV is on an increasing trend, with water supply, gas regulation, climate regulation, environmental purification and hydrological regulation being the highest value of individual services. Spatially, the total ESV showed that high levels were in the northwest, southwest and east, and low levels were in the northeast, west and central parts. At the same time, it shows a positive spatial correlation with a weakening trend. It is dominated by high–high cluster and low–low cluster. The change in the value of ecological services in forest land, grassland and water body have a greater impact on the change in total service value in the region, and was the main contributing and sensitive factor.