In-situ pressure coring technology is a responsible exploration technique for enhancing the efficiency and capacity of deep resources development. However, reliability issues in pressure sealing introduce significant uncertainty in field applications of this technology. This work presents a novel pressure sealing subsystem within the in-situ pressure-preserved coring system to overcome the inherent problem. The design concept and structure composition of the pressure sealing subsystem are described. To enhance pressure sealing reliability in real downhole conditions, the subsystem incorporates a dynamic sealing structure between the inner tube and the pressure bearing tube, and a close-fitting sealing face between the pressure controller and the bottom of the inner tube. Theoretical calculations and computational fluid dynamics (CFD) simulations were conducted to evaluate the mechanical behavior and fluid flow characteristics within the pressure sealing subsystem, determining the structural effects on performance. A smaller pump displacement during inner tube lifting and a moderate overflow hole diameter of 7 mm enhance the success rate of a sequence of mechanical actions required for the in-situ pressure sealing. Numerical, laboratory, and field tests were conducted to verify the service performance. Numerical analysis indicates that the particle settlement ratio in the novel structure is only 32% of that in the original design. In laboratory downhole circulation and drilling tests, the pressure sealing subsystem successfully maintained an in-situ pressure of 0.2 MPa at a depth of approximately 9–10 m. In field applications, a 1.95 m in-situ core sample was retrieved at 22 MPa from a depth of approximately 1970 m.