Terrestrial ecosystem carbon stock (TECS) is critical to socioeconomic development and ecosystem services and is jointly affected by land use and cover and climate change. However, the dynamics of long-term annual TECS levels in urban agglomeration remain largely unknown, and research mostly ignores the spatial heterogeneity of climate factors, compromising sustainable environmental management and land planning strategies. To this end, we integrated field observations of carbon density, land use, and climate factors to map the annual distribution of TECS and analyzed their spatiotemporal variations and policy implications in the urban agglomeration of the middle Yangtze River Basin in China from 1990 to 2020. The results showed that 43,855.47 km² of the land of the urban agglomeration changed from 1990 to 2020, accounting for 12.54% of the study area. The farmland and forest land area fluctuated and reduced, and the construction land area increased significantly. The increase in construction land was mainly from farmland and forest land. The TECS in urban agglomerations underwent a remarkable change, the overall trend fluctuated downward, and the maximum interannual variation was 1560 Tg. The transfer of construction land, farmland, forest land, shrubs, grassland, and other land mainly caused the change in carbon storage. Due to abnormal climate change, the urban agglomeration in some areas illustrated carbon storage with a spatially aggregated distribution. When considering the impact of climate change on carbon density, the TECS changes of land types other than forest land were found to be consistent with the area change but more significant due to climate change. The research results can provide reference data for regional land management policy formulation and realization of “dual carbon” goals.