Comfort is becoming an important issue that airlines use to differentiate themselves in a competitive market. Activity and posture, as an integrating external manifestation when passengers interact with the complex cabin system, could be used as an effective way to study passenger comfort. This study aims to analyze the passengers’ comfort perception based on activity and posture analysis performed by passengers on the flight. By recording and reconstructing the activities performed by passengers in a 2-hour simulated flight, the typical activities and the corresponding postures were identified through the video analysis software system MVTA. The passengers remained the greater part of the time performing the activity of sleeping and resting (34.3%), followed by using small electrical devices (32.7%) and reading (16.1%). The main postures in these activities were encoded and displayed in the elliptic structural diagram based on the variations in head, back, arms, and legs. The difficulties and constraints resulted from the seat and cabin were summarized by a questionnaire. Based on the analysis results, the suggestions about seat design, activity guidance, and arrangement were offered from the perspective of supportability, adjustability, affordance, and aesthetics, which would be applied in improving the passengers’ comfort and innovations in cabin and seat.