For the manned International Space Station, the more stringent requirements of the leakage performance of the rubber seal, which is located in it, is presented. The leakage is caused by the small leakage paths between contact pairs of the rubber seal. Therefore, it is necessary that the micro-leakage performance analysis between interfaces of the rubber seal is carried out. Based on this, the finite element model (FEM) of macroscopic contact performance analysis of the rubber seal is established first, and the macroscopic contact pressure and contact width of the rubber seal are calculated by the model. Then, the micro-leak path model is constructed based on the contact width and surface micro-structure, and the cross-sectional area of the micro-leakage path is obtained, when the contact pressure is acted on the model. Therefore, the fluid flow states in the micro-leakage path are determined, and the flow conductance and the seal leakage rate of rubber seal are all calculated. Finally, the micro-leakage model of the rubber seal is modified to improve the theoretical calculation accuracy by the experiment.