In order to explore spatial pattern of soil microbial biomass carbon (MBC) and its environmental driving forces in grassland surface layer (0?10 cm) and subsurface layer (10?20 cm) in the agro-pastoral transition zone in northern China, 456 soil samples (57 sample sites, two soil layers and four replicates) were selected for investigation from Inner Mongolia-Liaoning plain, Bashang on the north of Beijing, the northern foot of Yinshan Mountain and Ningxia-Shaanxi Loess Plateau. The results showed that the spatial pattern of MBC was consistent between the surface and subsurface layer, which increased with latitude and had no significant change with longitude and elevation. With the increase of degradation degree, the reduction of soil microbial biomass in the subsurface layer was smaller than that in the surface layer. The difference of soil pH between the two layers decreased with the degree of degradation. The difference in soil MBC between surface and subsurface soils was regulated by the change of soil pH. The smaller the difference in pH between the two soil layers, the smaller the difference in MBC. Climate, vegetation and soil factors could affect the spatial variation of MBC, and soil factors were the main driving forces for MBC at the regional scale. The main influencing factor of MBC was soil total carbon at surface layer, and soil total nitrogen at subsurface layer. In the context of climate change and the intensification of human activities, the results were useful for predicting the response of soil microorganisms at the regional scale and maintaining and restoring ecological functions of degraded grasslands.