Abstract: To improve the interference fit theory of multilayer cylinders under the centrifugal force and to obtain a better fit design scheme with the specified constraint conditions, the analytical formulas for stress analysis, deformation analysis and interference fit design were derived for a rotor with multi-layer rotating cylinders. Then, based on the above theory, with the minimum safety margin as a fitness function, the genetic algorithm was applied to optimize the nominal radii of the cylinders to maximize the minimum safety margin. According to the optimized and rounded nominal size, the finite element software (ANSYS Workbench) was used to establish a 3D model for the rotor of magnetic bearing to simulate the interference assembly process under the maximum interference, and the equivalent stress results by the finite element method and the analytical method were compared. The results show that the safety coefficient of rotating cylinders is increased by the proposed optimization method, which improves the security of the rotor. Also, the inner wall of the biggest cylinder in the model is the most dangerous torus, where the maximum equivalent stress by the finite element method is 18.72% higher than that by the analytical method, and the safety coefficient should be increased appropriately if only the analytical method is used for the equivalent stress prediction. Additionally, the analytic results are generally in a good agreement with the numerical results, therefore, the proposed analytical method can be used for the preliminary design or for the analysis on the interference fit of multilayer rotating cylinders so as to provide a theoretical guidance.