Multilayer interference fits are widely used in the machinery industry, and their performance is closely related to assembly quality. Press-fit is the primary assembly method. The pre-deformation of the previous assembly step can significantly affect the stress distribution (stress concentration) and pressing force in subsequent assembly steps. Therefore, the wear degree of the contact interface and the assembly quality of the components are affected by the assembly sequence. The multilayer thick-walled cylinder theory (MTCT) cannot realize the accurate analysis of stress distribution and pressing force during assembly. Therefore, this paper optimizes the MTCT considering the resistant force generated by the non-contact regions and proposes an assembly matrix generation algorithm and an assembly sequence optimization algorithm. Compared with the simulation analysis, the optimized theoretical model can be used for the analysis of the stress distribution and pressing force of multilayer interference components. The analysis accuracy is significantly improved compared with MTCT. The optimal assembly sequence determined by the proposed algorithm is consistent with simulation results.