Current and displacement stiffness are important parameters of axial magnetic bearing (AMB) and are usually considered as constants for the control system. However, in actual dynamic work situations, time-varying force leads to time-varying currents and air gap with a specific frequency, which makes the stiffness of appear decrease and even worsens control performance for the whole system. In this paper, an AMB dynamic stiffness model considering the flux variation across the air gap due to frequency is established to obtain the accurate dynamic stiffness. The dynamic stiffness characteristics are analyzed by means of the dynamic equivalent magnetic circuit method. The analytical results show that the amplitude of current and displacement stiffness decreases with frequency increasing. Moreover, compared with the stiffness model without considering the variation of flux density across the air gap, the improved dynamic stiffness results are closer to the actual results. Through the dynamic stiffness measurement method of AMB, experiments of AMB in magnetically suspended molecular pump (MSMP) are carried out and the experimental results are consistent with theoretical analysis results. This paper proposes the dynamic stiffness model of axial magnetic bearing considering the variation of flux density across the air gap, which improves the accuracy of the AMB stiffness analysis.