Large-scale magnetic levitation rotating machinery with multi-span rotors often require couplings to connect the rotors. Misalignment of the coupling will affect the stability of the rotor motion and even cause serious accidents. To make sure of the system stability， it is necessary to carry out research on the influence of coupling misalignment on the dynamic characteristics of the rotor. By equating the coupling misalignment to the rotational force exerted on the rotor， a mathematical model of the magnetic suspension rotor system with coupling misalignment is established. The axis track and vibration frequency spectrum of the rotor are simulated and analyzed based on this model， the relationship between the second-harmonic signal of rotor displacement and the rotation speed and the coupling misalignment is given. The experimental results show that at a certain speed， the amplitude of the second-harmonic displacement signal increases linearly by 1.53 μm for every 0.1 mm of coupling misalignment.