In this work, microstructural changes during creep of 9Cr-1Mo steel were characterized using ultrasonic and hardness measurements. The creep testing of this steel was firstly performed at 873 K under the initial stresses of 160 and 175 MPa, respectively. Then, eight specimens under each stress level were prepared by the interrupted tests to simulate various creep states. Subsequently, two parameters of ultrasonic attenuation and velocity were introduced by the ultrasonic testing. Experiment results showed that regardless of the stresses, the ultrasonic attenuation experiences a peak at about 20% of the creep rupture time and a minimum value at about 50%, showing a strong correlation between ultrasonic attenuation and the creep life fraction. In addition to a slight increase during the primary creep, the ultrasonic velocity exhibits a significant change during the tertiary creep. Based on the analysis of microstructure changes, it indicated that the ultrasonic attenuation is significantly sensitive to the dynamic changes of the dislocation. In contrast, the velocity is mainly related to the evolution of precipitates. The Vickers hardness decreases monotonously during the creep process and presents a linear relation with the inverse of average diameter of M23C6 carbides during the secondary and tertiary creep. Accordingly, it suggested that the present ultrasonic and hardness measurements provided a convenient tool to evaluate the microstructure evolution during creep degradation of heat-resistant steels.