Aims Multiple studies have revealed that soil organic carbon (SOC) continues to accumulate as forests age, old-growth forests had higher SOC content. Microbial carbon use efficiency (CUE) and its accurate assessment is crucial for understanding C cycling. Most previous studies were based on a single approach, which limits our ability to cross-compare CUE across studies and fully utilize the complementary information gained. However, whether the response of microbial CUE to environmental change and the relationship with SOC follows a consistent trend under different methods remain unclear. Methods Therefore, we explore microbial CUE in long-term subalpine forests (middle-aged and old-growth forests, MF and OF) under three approaches and evaluated how microbial CUE response to forest age and soil layer and how it relates to SOC. Results The choice of methods significantly affected microbial CUE, with mean value was C-13 (0.60) > O-18 (0.38) > SM (0.26). Irrespective of methods, the microbial CUE in OF is higher than in MF and it declined with soil depth. Microbial CUE increased from MF to OF by 8.1%, 29.2% and 23.9%, and decreased from the O to B layer by 6.8%, 18.9% and 127.3% measured by the C-13, O-18 and SM methods. And microbial CUE was positively related to MBC and SOC content under three methods, higher soil microbial CUE supported higher SOC in old-growth forests. Conclusion Microbial CUE response to forest age and soil layer was independent of approaches, but the extent was varied. And our results provide the accurate assessment of microbial CUE and important implications for strengthening SOC management in mature forests.