An accurate modal analysis of the stepped rotor assembly is significant in the design process of rotors. The transfer matrix method (TMM) is widely used in the modal analysis of the rotor. However, the influences of the step and the assembling modes are not considered in the traditional TMM. In this paper, a modified transfer matrix method is presented for the modal calculation of the rotor in the turbomolecular pump. The stiffness reduction of stepped segments and the effect of assembled components are considered in this modified method. First of all, the traditional transfer matrix model is built. Then, the stepped segments of the rotor are changed into conical segments and the lumped mass model of conical segments is calculated. Next, the conicities of conical segments are analyzed and relations between conicities and dimensions of the rotor are discovered. Finally, the stiffness factors which can describe the effects of different assembling modes are introduced and optimized. The optimized stiffness factors are analyzed and explained from the perspective of contact stress. The modal of the stepped rotor assembly could be computed with high accuracy by using this method.