AbstractSoil microbial biomass is a key soil driver in the management of ecosystem functioning. However, little is known about the spatial distribution of microbial biomass in frequently disturbed areas, such as agro‐pastoral ecotones. In this study, we sampled soil from natural grasslands across 56 sites in the agro‐pastoral ecotone of northern China at 0–10 and 10–20 cm soil depth. We measured microbial biomass carbon (MBC), nitrogen (MBN), and phosphorus (MBP) and possible drivers of responses. Both MBC and MBP showed a significant positive relationship with latitude at the regional scale, whereas MBN showed a non‐significant latitudinal pattern, independent of soil depth. The major influential factors for the latitudinal pattern of both MBC and MBN were soil total carbon content (STC), soil total nitrogen content (STN), and soil moisture, while the latitudinal pattern of MBP was influenced by STN, soil pH and mean annual temperature. Furthermore, MBC, MBN, and MBP decreased with increasing soil depth by 37.4%, 32.8%, and 21.5%, respectively. Interestingly, when microbial biomass was normalized by STC, STN, and soil total phosphorous content (STP), MBC/STC still significantly declined, whereas MBN/STN showed no significant difference and MBP/STP significantly increased with soil depth. The influential factor for the depth patterns of MBC, MBN, and MBP was soil substrate (as indicated by STC, STN, and STP). The findings of this study are helpful in understanding the spatial pattern of microbial biomass and its influencing factors in frequently disturbed areas.