In practical engineering applications, most connection interfaces require lubrication. The contact stiffness of the lubrication interface can affect the static and dynamic characteristics of the interface, thus affecting the performance of the mechanical system, such as vibration, fatigue, and wear characteristics. Therefore, the measurement of lubrication contact stiffness is very important for the analysis and optimization of mechanical system performance. However, measuring the stiffness of the lubrication interface is a significant challenge. In this paper, a mixed lubrication contact stiffness model considering asperity interactions is proposed, which assumes the lubrication rough interface as an equivalent thin layer. The proposed model was compared with the lubrication contact model that does not consider the asperity interactions. Based on the microscopic contact behavior of the contact interface, the relationship between the mechanical parameters of the virtual thin layer and the contact pressure was analyzed. Finally, the predicted results of interface stiffness are verified by published theoretical and experimental results. The results show that the proposed model can more reasonably describe the actual contact behavior of the rough lubrication interface and give the variation law of the mechanical parameters of the virtual thin layer.