The sealing performance is a critical characteristic of the stop valve used in an 11000-m manned submersible, while it is a challenge when the pressure difference at the valve port reaches 120 MPa. Previous studies have mainly focused on the effect of surface microtopography, such as the surface roughness, on sealing performance. They believed that rougher surfaces will result in more severe leaks. However, in the sealing experiments of two stop valves with different materials and surface roughness in this paper, the relationship between surface roughness and sealing performance is inconsistent with existing research. There should be another factor that affects the sealing performance of the stop valves more strongly. So a new sealing principle considering the roundness error is proposed. The sealing force, which is determined by the environmental pressure, creates radial deformation on the valve port. The radial deformation of the valve seat is analyzed by a validated finite element analysis (FEA) at various environmental pressures. Then the critical environmental pressure at which the leak stops is obtained when the radial deformation is greater than the roundness error, which is measured. The results show that the critical environmental pressure obtained by FEA is very close to that obtained by experiment. So the consideration of the roundness error of the valve seat is accurate and helpful to the research on the sealing performance of the ultra-high pressure stop valves, and the sealing performance of the ultra-high pressure stop valve can be easily evaluated by FEA.