The hydrogen and carbon isotope ratios of CH4 emitted from one bonfire and two laboratory combustions of agricultural residues were determined in this study as −196 to −262‰ for δD and −19.9 to −35.1‰ for δ13C. The isotopic difference between CH4 emitted from biomass burning and fuel biomass, that is, the apparent isotopic fractionation occurring during combustion (ɛBurn), was within −101 to −174‰ for ɛBurnD and +6.9 to −8.6‰ for ɛBurn13C. That difference varied according to burning conditions: flaming and smoldering. Variation in ɛBurn is correlated with combustion efficiency (CE), defined here as the ratio of emitted CO2 to the sum of emitted CO2 and CO. In light of the previously reported global distributions of CE, the implied ɛBurn values for global biomass burning sources were −121‰ for ɛBurnD and +2.2‰ for ɛBurn13C. Using the relationship between ɛBurn and CE and global distributions of CE and isotopic ratios of fuel biomass, we estimated the global isotopic signature for biomass burning sources as −169‰ for δD and −23.6‰ for δ13C.