The desert steppe serves as a transitional zone between grasslands and deserts, and long-term monitoring of aboveground biomass (AGB) in the desert steppe is essential for understanding grassland changes. While AGB observation techniques based on multisource remote-sensing data and machine-learning algorithms have been widely applied, research on monitoring methods specifically for the desert steppe remains limited. In this study, we focused on the desert steppe of Inner Mongolia, China, as the study area and used field sampling data, MODIS data, MODIS-based vegetation indices (VI), and environmental factors (topography, climate, and soil) to compare the performance of four commonly used machine-learning algorithms: multiple linear regression (MLR), partial least-squares regression (PLS), random forest (RF), and support vector machine (SVM) in AGB estimation. Based on the optimal model, the spatial–temporal characteristics of AGB from 2000 to 2020 were calculated, and the driving forces of climate change and human activities on AGB changes were quantitatively analyzed using the random forest algorithm. The results are as follows: (1) RF demonstrated outstanding performance in terms of prediction accuracy and model robustness, making it suitable for AGB estimation in the desert steppe of Inner Mongolia; (2) VI contributed the most to the model, and no significant difference was found between soil-adjusted VIs and traditional VIs. Elevation, slope, precipitation, and temperature all had positive effects on the model; (3) from 2000 to 2020, the multiyear average AGB in the study area was 58.34 g/m2, exhibiting a gradually increasing distribution pattern from the inner region to the outer region (from north to south); (4) from 2000 to 2020, the proportions of grassland with AGB slightly and significantly increasing trend in the study area were 87.08% and 5.13%, respectively, while the proportions of grassland with AGB slightly and significantly decreasing trend were 7.76% and 0.05%, respectively; and (5) over the past 20 years, climate change, particularly precipitation, has been the primary driving force behind AGB changes of the study area. This research holds reference value for improving desert steppe monitoring capabilities and the rational planning of grassland resources