Soil microorganisms and their extracellular enzymes are key determinants of the biogeochemical cycles of carbon (C), nitrogen (N), and phosphorus (P). The objective of this study is to characterize the distribution patterns of soil microbial biomass and soil extracellular enzyme activity along different elevations and soil horizons within a typical sub-basin on the Tibetan Plateau. Additionally, this paper aims to identify the major nutrient-limited conditions that affect these distribution patterns. In this study, soil samples were collected in August 2021 from four layers of soil occurrence (layer A: humus layer, layer E: leached layer, layer B: sediment layer, and layer C ∶ parent material layer) at five different elevations (4900 m, 5000 m, 5100 m, 5200 m, and 5300 m) in the Gokyo Gangri glacier sub-basin on the Tibetan Plateau. The soil samples were analyzed to determine their basic physical and chemical properties, soil microbial biomass, soil extracellular enzyme activity, and microbial carbon use efficiency. The results showed that: 1) the elevation differences of carbon, nitrogen and phosphorus of microbial biomass varied, with the deepening of soil layer, the carbon, nitrogen and phosphorus of microbial biomass changed less with elevation. At the same time, the microbial biomass tended to decrease significantly (P<0.05) with the deepening of the soil layer between each elevation. 2) The elevation variation pattern of the four enzyme activities varied, but the overall trend of increasing with elevation was more obvious in the surface layers (A and E) and decreased significantly (P<0.05) with the deepening of the soil layer. 3) The soil microorganisms in this area were limited by both carbon and phosphorus. The deeper the soil layer, the higher the limitation, and the higher the elevation, the stronger the C limitation, but the lower the P limitation. 4) Changes in soil vertical profile had a significant effect on changes in microbial Carbon use efficiency(CUE)(P<0.01). There were pronounced disparities in soil microbial biomass and extracellular enzyme activity observed among various altitudes and soil layers within a typical glaciated sub-basin on the Tibetan Plateau. These disparities are likely attributed to variations in soil nutrient composition and pH levels, which significantly influence the stoichiometry of extracellular enzymes. Overall, the soil nutrient content of typical glacial sub-basins on the Tibetan Plateau influenced the distribution characteristics of soil microbial biomass and extracellular enzyme activity between altitudes and soil horizons. The study provides a data base and scientific basis for an in-depth understanding of soil nutrient cycling in glacial sub-basins on the Tibetan Plateau.