Threatened animals respond with appropriate defensive behaviors to survive. It has been accepted that midbrain periaqueductal gray (PAG) plays an essential role in the circuitry system and organizes defensive behavioral responses. However, the role and correlation of different PAG subregions in the expression of different defensive behaviors remain largely unexplored. Here, we designed and manufactured a microelectrode array (MEA) to simultaneously detect the activities of dPAG and vPAG neurons in freely behaving rats. To improve the detection performance of the MEAs, PtNP/PEDOT:PSS nanocomposites were modified onto the MEAs. Subsequently, the predator odor was used to induce the rat’s innate fear, and the changes and information transmission in neuronal activities were detected in the dPAG and vPAG. Our results showed that the dPAG and vPAG participated in innate fear, but the activation degree was distinct in different defense behaviors. During flight, neuronal responses were stronger and earlier in the dPAG than the vPAG, while vPAG neurons responded more strongly during freezing. By applying high-performance MEA, it was revealed that neural information spread from the activated dPAG to the weakly activated vPAG. Our research also revealed that dPAG and vPAG neurons exhibited different defensive discharge characteristics, and dPAG neurons participated in the regulation of defense responses with burst-firing patterns. The slow activation and continuous firing of vPAG neurons cooresponded with the regulation of long-term freezing responses. The results demonstrated the important role of PAG neuronal activities in controlling different aspects of defensive behaviors and provided novel insights for investigating defense from the electrophysiological perspective.