Knowledge about the elevational patterns of soil microbial biomass and communities can facilitate accurate prediction of the responses of soil biogeochemical processes to climate change. However, previous studies that have considered intra- and inter-annual variations have reported inconsistent results on the one hand, and they have paid little attention to the effect of soil layer on the other hand. We, therefore, conducted a 4-year in situ soil core incubation experiment along a 2431-m elevational gradient across the dry valley shrubland, valley-montane ecotone forest, subalpine coniferous forest, alpine coniferous forest, and alpine meadow in an ecologically fragile alpine-gorge region on the eastern edge of the Qinghai-Tibetan Plateau. Soil microbial biomass and community composition in the organic and mineral layers were measured using the phospholipid fatty acids (PLFA) method at five critical periods each year. Our results indicated that soil microbial biomass in the organic layer was the highest in the subalpine coniferous forest, followed by the alpine meadow, alpine coniferous forest, and valley-montane ecotone forest. In contrast, soil microbial biomass in the mineral layer was significantly higher in the alpine meadow than in the other sites. Soil microbial biomass exhibited differential seasonal fluctuations at different elevations, resulting in their elevational patterns being strongly intra-annual and inter-annual dependent. Our results revealed that elevation and seasonality significantly affected soil microbial communities. Seasonality had a more substantial effect than elevation on soil microbial communities during the first three years of incubation, whereas the relative importance of seasonal and elevational effects on microbial communities was reversed in the organic layer with incubation time. These results are mainly attributed to the magnitude and direction of effect of environmental variables on soil microbial biomass and communities vary with elevation, soil layer, and sampling time. Briefly, the elevational patterns and dominant factors of soil microbial biomass and communities have intense soil layer and temporal specificity, implying that differential responses of soil biochemical processes to climate change might be observed at different elevations.