It is crucial to accurately characterize the static and dynamic properties of seals for the design of deep-sea equipment. This study investigated the movement resistance of O-rings under high pressure (up to 30 MPa) and low temperature (2 °C). Numerical simulation models were set up to investigate the effects of material properties and seal structure dimensions on the reciprocating friction and installation force of O-rings. Furthermore, a novel resistance testing rig was developed to facilitate the measurement of reciprocating friction and installation force of O-rings under high pressure and low temperature. The results of this study indicate that nitrile rubber (NBR) has a reduced sensitivity to temperature variations, and the hardening of the material due to the low temperatures encountered in deep-sea environments does not substantially increase its movement resistance. Conversely, fluororubber (FKM) exhibits superior static sealing reliability. We optimized the sealing structure of the pressure-maintaining trapping instrument (PMTI) and successfully conducted field tests at the South China Sea. The results of this study may serve as a valuable reference for the sealing design of deep-sea equipment.