ABSTRACTWith the widespread and ever‐increasing application of microwave technology in our daily lives, the potential adverse effects on biological systems from exposure to microwaves are extremely interesting based on thermal and nonthermal effects, especially nonthermal effects on the nervous system. To real time and dynamically elucidate the diversity of reactive oxygen species (ROS) in rat pheochromocytoma (PC12) cells using a real‐time‐pulsed microwave device combining laser confocal microscopy (LSCM). A LSCM system was designed, and its physical properties, including irradiation cavity simulation, field intensity distribution, and average specific absorption rate (SAR), were analyzed and a stable field intensity distribution as well as a precise average SAR were observed. Subsequently, the diversity of ROS in PC12 cells under pulsed microwave exposure was determined by LSCM online. Additionally, the total ROS in PC12 cells was determined by flow cytometry after pulsed microwave exposure. The ROS signal in single PC12 cell was rapidly quenched as the irradiation time was extended, whereas the total ROS showed no changes in flow cytometry after microwave exposure. This indicates that the change of intracellular ROS in PC12 cells by microwave exposure is a real‐time and dynamic process, and provides a significant reference for studying the biological effects of microwaves. © 2015 Wiley Periodicals, Inc. Microwave Opt Technol Lett 57:2395–2400, 2015