Abstract: Guizhou province is located in the typical karst landform area with a fragile ecology. It is of significance to analyze the influence of land use change caused by multiple driving factors on the value of regional ecosystem service for ecological construction and land management. Based on GlobeLand30 land use/cover dataset, grain production and price, we calculated the dynamic degree of land use, degree of utilization, transfer matrix and spatial autocorrelation index and corrected the service value coefficient of ecosystem in Guizhou Province. The change of land use and ecosystem service value in Guizhou Province from 2000 to 2020 was analyzed, and the influence and interaction of driver factors were quantified by geographical detectors. This study found that: (1) During the study period, forest land and grassland decreased by 1 148.79 km2 and 1 600.36 km2, respectively, while water and artificial surface increased by 496.54 km² and 2 315.81 km², respectively. The comprehensive land use dynamic degree reached 45.16%, and the artificial surface dynamic degree was 32.75%; The spatial displacement process of cultivated land, forest and grassland, and artificial surface was significant, and there was a trade-off between artificial surface and cultivated land; (2) The high value of land use degree of utilization mainly revolved around the distribution of urban areas in Guiyang, Anshun, and Zunyi, and the distribution of land use exhibited spatial clustering, with global Moran′s I values greater than 0. The artificial surface exhibited significant high-high clustering (H-H) characteristics; (3) Population and road network had a sustained impact on the spatial pattern of land use, while precipitation was associated with the distribution of arable land and forest land. The driving factors had a synergistic effect on the spatial pattern of land use; (4) In the past 20 years, the value of ecosystem services in Guizhou Province decreased first and then increased. The value of forest and grass ecosystem services accounted for over 80% of the total value, and regulation and support services were the main types of ecosystem services. In 29 counties in Guizhou Province, the value of ecosystem services shifted from loss to gain. For future ecological construction, it is necessary to strengthen the construction and management of forest and grass ecosystems.