【Objective】Microbial carbon use efficiency (CUE) is a key metric for quantifying the proportion of absorbed carbon converted into microbial biomass, and plays a critical role in understanding microbial metabolism and soil carbon turnover processes. However, the different methods used for determining CUE present dissimilarities, which affects the reconciliation of global data. Therefore, this study aimed to evaluate the differences and connections among diverse approaches for characterizing CUE.【Method】This study focused on typical red soils with fertility gradients from a long-term fertilization experiment station, employing three methods—13C tracing (13C-glucose), 18O tracing (18O-H2O), and stoichiometric modeling, to characterize CUE, and compare the factors influencing these methods.【Result】The results showed that the CUE measured by the 13C tracing method (0.63-0.81) was significantly higher than those obtained by the 18O tracing method (0.26-0.52) and the stoichiometric modeling approach (0.35-0.55) (P < 0.05). Both the microbial growth rate and respiration rate measured by the ¹³C tracing method were significantly higher than those by the ¹⁸O tracing method (P < 0.05). Soil pH, the structure of dissolved organic matter (DOM) (Specific UV Absorbance at 254 nm, SUVA254; Humification Index, HIX), and the intensity of microbial resource limitation (vector length and vector angle of enzyme stoichiometry) were identified as the main factors influencing CUE. The CUE characterized by all three methods showed a significant positive correlation with pH and SUVA254 (P < 0.05). However, only the CUE characterized by the stoichiometric method showed a significant positive correlation with HIX (P < 0.05). The microbial growth rates measured by the ¹³C and ¹⁸O tracing methods both increased significantly with soil pH (P < 0.05). Moreover, microbial respiration rates measured by the ¹⁸O tracing method showed a significant positive correlation with soil pH (P < 0.05), whereas those measured by the ¹³C tracing method did not correlate significantly with soil pH (P > 0.05). The CUE characterized by the 13C tracing method and stoichiometric modeling decreased significantly with increasing microbial carbon limitation intensity (P < 0.05), whereas the CUE measured by the 18O tracing method showed no significant correlation with microbial resource limitation intensity (P > 0.05).【Conclusion】Therefore, it is recommended that biogeochemical models should account for the differences in CUE and its driving factors under diverse characterization methods. This will permit the accurate prediction of the responses of microorganisms to carbon sources.