The Qinghai–Tibetan Plateau is the highest plateau in the world and is sensitive to climate change. The dynamics of soil enzyme activities and microbial communities are good indicators of alpine biochemical processes during warming. We collected topsoil (0–10 cm) and subsoil (10–20 cm) samples at altitudes of 3200–4000 m; determined the activities of β-1,4-glucosidase (BG), cellobiohydrolase (CBH), β-1,4-N-acetyl-glucosaminidase (NAG) and acid phosphomonoesterase (PME); and performed Illumina 16S rRNA high-throughput sequencing. We found that the soil carbon (total organic carbon and dissolved organic carbon) and nitrogen (total nitrogen and dissolved organic nitrogen) fluctuated with altitude in both the topsoil and subsoil, whereas the dissolved phosphorus continuously decreased with the increasing altitude. BG and CBH decreased from 3200 to 3600 m and increased from 3800 to 4000 m, with the lowest levels occurring at 3600 m (topsoil) and 3800 m (subsoil). NAG and PME showed similar fluctuations with altitude, with the highest levels occurring at 3400 m and 4000 m in both the topsoil and subsoil. Generally, the altitudes from 3600 to 3800 m were an ecological transition belt where most of the nutrients and enzyme activities reached their lowest levels. All of the alpine soils shared similar dominant phyla, including Proteobacteria (32.7%), Acidobacteria (30.2%), Actinobacteria (7.7%), Bacteroidetes (4.4%), Planctomycetes (2.9%), Firmicutes (2.3%), Gemmatimonadetes (2.0%), Chloroflexi, (1.2%) and Nitrospirae (1.2%); Gemmatimonadetes and Verrucomicrobia were significantly affected by soil depth and Planctomycetes, Firmicutes, Gemmatimonadetes, Nitrospirae, Latescibacteria and Armatimonadetes were significantly affected by altitude. In addition, nutrient availability, enzyme activity and microbial diversity were higher in the topsoil than in the subsoil, and they had more significant correlations in the subsoil than in the topsoil. Our results provide useful insights into the close linkages between soil nutrient cycling and microbial activities on the eastern Qinghai–Tibetan Plateau, and are of great significance for further assessing the long-term impact of environmental changes in the alpine ecosystems.