O-ring seal structure is widely used in the weapon systems of air and ship, and its sealing performance di-rectly affect the operational mission of the weapon systems. The existing design methods of O-ring seal do not suffi-ciently consider the effects of manufacturing tolerance, medium pressure fluctuation, etc., on the reliability of O-ring sealing performance, and cannot give quantitative value of reliability index. In this paper, the stress-strength interference theory is employed to establish a limit state function of O-ring seal structure considering the influencing factors men-tioned above, and corresponding calculation method of reliability is proposed. Then, reliability of the O-ring seal struc-ture for an underwater vehicle is calculated by the software FREET. This study may provide a reference for design of O-ring seal structure.