Aromatic nitro compounds are an important class of basic chemical raw materials, while traditional nitration methods may cause safety accidents and environmental pollution due to strong exothermicity and presence of sulfuric acid. In this work, aiming to alleviate these issues, the nitration of toluene was conducted in a microchannel reactor with nitric acid and acetic anhydride. The effect of temperature, molar ratio of nitric acid to toluene, mass fraction of acetic anhydride, and residence time on the conversion and selectivity of reaction were systematically investigated, and response surface methodology was used to optimize the process. A mononitrotoluene yield of 99.21% can be achieved under optimum condition. Meanwhile, in order to better understand the exothermic behavior, toluene nitration by HNO3-AC2O was investigated in the semi-batch calorimeter. Finally, the optimized process was scaled up and effect of volumetric flow rate was investigated. At the selected maximum volumetric flow rate, the overtemperature of the system was 10.9°C with a 98.3% yield of mononitrotoluene. Continuous flow mode had a space-time yield nearly two orders of magnitude greater than semi-batch mode. This work can provide guidance for safe, efficient and green synthesis of mononitrotoluene.