With the improvement of modern aviation equipment manufacturing technology, there are relatively few failures due to the unreliability of the aircraft. However, human factors which resulted in air crashes and unsafe events are raised. In this paper, for many typical accident scenarios of a particular plane, the flight simulation verifications of the pilot’s workload and behavior are carried out on the certain transport category airplane, namely the six-degrees-of-freedom full-motion flight simulator. The subjective and physiological evaluation methods combine to analyze the human factors of pilots in the sudden typical accident scene during a flight mission. The study uses eye trackers, professional heart rate monitors, cameras, and other equipment to collect the pilot’s physiological information during the flight mission, and allows the pilots to fill in the subjective evaluation scale, establishing a subjective and objective evaluation index system. Thus, the human factors of pilots in typical fault situations are analyzed. The analysis shows the combined personal and accurate evaluation method, with the test equipment and environment proposed by this paper being feasible for the human factor evaluation in the accident or incident of transport category airplanes. It will benefit aviation stakeholders in determining the proper action to decrease the workload to an acceptable level.