[Objective] The deterioration of grasslands has become one of the major environmental problems in China. Both soil microbial biomass and extracellular enzyme activity are important indices, influencing soil nutrients and environmental quality. We aim to reveal the effect of grassland deterioration on soil microbiological characteristics along a depth profile. [Methods] The Inner Mongolia steppe was selected due to its typical deterioration features. We took samples at different depths (0 to 100 cm) from four grasslands including mature grassland, moderately deteriorated grassland, severely deteriorated grassland and extremely deteriorated grassland. We measured soil microbial biomass and the activity of extracellular enzymes involved in carbon and nitrogen cycling. [Results] Microbial biomass and enzyme activity at the topsoil layer showed the same trend in different deteriorated grasslands: mature grassland > moderately deteriorated grassland > severely deteriorated grassland > extremely deteriorated grassland. The differences between the 10?20 cm and topsoil in microbial biomass and enzyme activity were reduced with deterioration, and the microbial biomass and enzyme activity at the 10?20 cm layer were higher than that at the topsoil in the extremely deteriorated grassland. [Conclusion] Microbial biomass and enzyme activity at the topsoil layer decreased with grassland deterioration. When the deterioration of grasslands became more severe, the differences in soil microbiological characteristics between the surface and the 10?20 cm soil were less. These results provide a new method for assessing the degradation of grasslands, and thus offer important theoretical bases for the restoration of degraded temperate grassland.