Alpine ecosystems are important terrestrial carbon (C) pools, and microbial decomposers play a key role in litter decomposition. Microbial metabolic limitations in these ecosystems, however, remain unclear. The objectives of this study aim to elucidate the characteristics of microbial nutrient limitation and their C use efficiency (CUE), and to evaluate their response to environmental factors. Five ecological indicators were utilized to assess and compare the degree of microbial elemental homeostasis and the nutrient limitations of the microbial communities among varying stages of litter decomposition (L, F, and H horizon) along an altitudinal gradient (2800, 3000, 3250, and 3500 m) under uniform vegetation (Abies fabri) on Gongga Mountain, eastern Tibetan Plateau. In this study, microorganisms in the litter reached a strictly homeostatic of C content exclusively during the middle stage of litter decomposition (F horizon). Based on the stoichiometry of soil enzymes, we observed that microbial N- and P-limitation increased during litter degradation, but that P-limitation was stronger than N-limitation at the late stages of degradation (H horizon). Furthermore, an increase in microbial CUE corresponded with a reduction in microbial C-limitation. Additionally, redundancy analysis (RDA) based on forward selection further showed that microbial biomass C (MBC) is closely associated with the enzyme activities and their ratios, and MBC was also an important factor in characterizing changes in microbial nutrient limitation and CUE. Our findings suggest that variations in MBC, rather than N- and P-related components, predominantly influence microbial metabolic processes during litter decomposition on Gongga Mountain, eastern Tibetan Plateau.