The Eurasian steppe is the largest steppe region in the world and is an important part of the global grassland ecosystem. The eastern Eurasian steppe has favorable hydrothermal conditions and has the highest productivity and the richest biodiversity. Located in the arid and semi-arid region, the eastern Eurasian steppe has experienced large-scale grassland degradation due to dramatic climate change and intensive human activities during the past 20 years. Hence, accurate estimation of aboveground biomass (AGB, gC m-2) and belowground biomass (BGB, gC m-2) is necessary. In this study, plenty of AGB and BGB in-situ measurements were collected among dominated grassland types during summer in 2013 and 2016-2018 in the eastern Eurasian steppe. Vegetation indices from the Moderate Resolution Imaging Spectroradiometer (MODIS), Digital Elevation Model (DEM) and climate variables were chosen as independent variables to establish predictive models for AGB and BGB with random forest (RF). Both AGB (R2 = 0.47, MAE = 21.06 gC m-2, and RMSE = 27.52 gC m-2) and BGB (R2 = 0.44, MAE = 173.02 gC m-2, and RMSE = 244.20 gC m-2) models showed acceptable accuracy. Then the RF models were applied to generate spatially explicit AGB and BGB estimates for the study area over the last two decades (2000-2018). Both AGB and BGB showed higher values in the Greater Khingan Mountains and decreased gradually to the east and west sides. The mean values for AGB and BGB were 62.16 gC m-2 and 531.35 gC m-2, respectively. The climatic factors were much more important in controlling biomass than anthropogenic drivers, and shortage of water and raising temperature were the main limiting factor of AGB and BGB, respectively, in the peak growth season. These findings provide scientific data for the scientific management of animal husbandry and can contribute to the sustainable development of grassland ecology in the eastern Eurasian steppe.