The relationship framework between electroencephalogram (EEG) signals and subjective perception during pilots’ missions was established to improve pilots’ training efficiency and flight safety. Primarily, this study constructs a real flight scene through virtual reality (VR) and then obtains EEG data in simulated scenes. Researchers use VR technology to build a mission simulation room and then acquire EEG data from participants wearing EEG acquisition devices in the simulated room. The experimental process is divided into flight simulation and a questionnaire survey. Based on the participants’ EEG analysis, the researchers verified the changes of β rhythm under a high-difficulty operation mission. In addition, this study infers the mechanism of affecting pilots’ mental workload under high-difficulty operation by analyzing the correlation between subjective questionnaire results and β rhythms. The results showed that in the context of pilots performing flight missions in the aircraft space environment, the pilots’ mental load had the most excellent rhythmic relationship with the regions representing β rhythm. Therefore, a comprehensive experimental framework constructed in this study based on virtual simulation space to analyze the relationship between EEG and NASA-TLX subjective questionnaire provides a set of more accurate reference information for the design of a pilot training system based on pilot training efficiency and flight safety.