To accurately assess how increased global nitrous oxide (N2O) emission has affected the climate system requires a robust estimation of the preindustrial N2O emissions since only the difference between current and preindustrial emissions represents net drivers of anthropogenic climate change. However, large uncertainty exists in previous estimates of preindustrial N2O emissions from the land biosphere, while preindustrial N2O emissions on the finer scales, such as regional, biome, or sector scales, have not been well quantified yet. In this study, we applied a process-based Dynamic Land Ecosystem Model (DLEM) to estimate the magnitude and spatial patterns of preindustrial N2O fluxes at the biome, continental, and global level as driven by multiple environmental factors. Uncertainties associated with key parameters were also evaluated. Our study indicates that the mean of the preindustrial N2O emission was approximately 6.20TgNyr−1, with an uncertainty range of 4.76 to 8.13TgNyr−1. The estimated N2O emission varied significantly at spatial and biome levels. South America, Africa, and Southern Asia accounted for 34.12, 23.85, and 18.93%, respectively, together contributing 76.90% of global total emission. The tropics were identified as the major source of N2O released into the atmosphere, accounting for 64.66% of the total emission. Our multi-scale estimates provide a robust reference for assessing the climate forcing of anthropogenic N2O emission from the land biosphere