Mixed lubrication interfaces are widespread in engineering. The measurement of contact stiffness is a challenge for device performance evaluation. The ultrasonic reflection method is an effective method, but the difficulty of accurate construction of the acoustic model limits the measurement accuracy. In this study, an improved acoustic model is proposed to analyze the contact stiffness of the mixed lubrication interface. The model is constructed using a quasi-static spring model, virtual material model, statistical microcontact model, and multilayer acoustic model. The mixed lubrication interface is equivalent to a homogeneous and isotropic virtual material layer. Then, the mechanical, geometric, and acoustic parameters of the virtual layer are determined by introducing thickness coefficients. The contact stiffness is obtained with the proposed model and compared with the quasi-static spring model and the virtual material layer model. The reflection coefficient of the interface can also be calculated using a multilayer acoustic model based on the calculated parameters. The proposed model is verified by comparing the predicted reflection coefficients with the published experimental results.