Canopy gaps are a key component of the disturbance regime and old-growth character in subalpine coniferous forests. Despite the importance of canopy gaps in the dynamics and management of subalpine forests, limited information is available on the temporal dynamics of microbial pools and their roles in the responses of soil organic matter and nutrient flux to canopy gaps. This study tests the hypothesis that gap creation facilitates soil organic carbon (C) retention by soil microbial biomass in the organic horizon. Based on the degree of decomposition, the organic horizon can be divided into fresh litter layer (LL), fragmented litter layer (FL) and humified litter layer (HL) components; these layers represent different stages of litter decomposition. We examined the microbial biomass carbon (MBC), microbial biomass nitrogen (MBN) and microbial biomass phosphorus (MBP) from the gap center to closed canopy in a subalpine Minjiang fir (Abies faxoniana) forest during different seasons. Gap creation promoted soil organic C retention by soil microbial biomass in the organic horizon. Storage of total organic carbon (TOC), total nitrogen (TN) and total phosphorus (TP) accumulated in the HL. Moreover, gap creation accelerated C, nitrogen (N) and phosphorus (P) releases from the LL but inhibited C, N and P outputs from the HL. Microbial biomass in the LL responded rapidly and sensitively to canopy gaps. The most important factors affecting microbial biomass in the organic horizon were soil temperature and snow cover, which are governed by the canopy gap. Microbial pools and their stoichiometry were significantly and positively correlated with TOC. A thorough comprehension of the spatio-temporal traits of microbial biomass pools in the organic horizon under gap creation could aid the regeneration of protected subalpine fir forest on the eastern Tibetan Plateau.