The large deformation rubber cylinder is one of the most important core components of the pipeline plugging robot. Its function is mainly to isolate the oil and gas in the pipeline during pipeline repair and maintenance operations. In field use, the problems of rubber cylinder seal failure and tear failure are more prominent. In order to clarify the above reasons and solve practical engineering problems, this paper studied the effects of different rubber materials, different rubber cylinder axial lengths, radial lengths, inclined edges and other structural parameters on the static sealing characteristics of the pipeline plugging robot. Based on the high elasticity and large deformation characteristics of the rubber material, the multi-stage deformation mechanics analysis of the rubber cylinder was carried out. The uniaxial tensile-compression test was carried out on the rubber material, and the constitutive relation parameters of the rubber material were obtained. The finite element calculation model for the analysis of the sealing characteristics of the rubber cylinder of the pipeline plugging robot was established, and the optimal scheme of the key structural parameters of the rubber cylinder was obtained through the multi-factor analysis method. An indoor test was designed to further determine the optimal rubber cylinder material. It showed that the contact stress between rubber cylinder and pipe wall and the value of the sealing performance coefficient were the key parameters for evaluating the static sealing performance of the pipeline plugging robot. The Mises stress concentrated on the compressed surface of the rubber cylinder directly affected the deformation and damage of the rubber cylinder. Reducing the Mises stress here was beneficial to improve the service life of the pipeline plugging robot. Under the same load, as the hardness of the rubber cylinder material increased, the sealing performance of the rubber cylinder was worse. As the axial length of the rubber cylinder soared, the sealing performance of the rubber cylinder was better, but the Mises stress on the compressed surface of the rubber cylinder was greater. As the radial thickness of the rubber cylinder decreased, the sealing performance of the rubber cylinder was better, but the Mises stress on the compressed surface of the rubber cylinder was greater. With the increase of the rubber cylinder inclined angle, the sealing performance of the rubber cylinder first increased and then weakened, and the Mises stress on the compressed surface of the rubber cylinder first decreased and then increased. After the comparative analysis of the sealing effect of the rubber cylinder, the optimal rubber cylinder structure was obtained as the axial length of 180 mm, the radial thickness of 55 mm, the inclined edge angle of 28°, and the rubber material hardness of 85 HA. The results of this paper had important reference significance for the development of high-performance, long-life, and high-reliability pipeline plugging robots.