Objective This paper aimed to study the effect of prosthetic alignment on the internal contact mechanical characteristics of intact knee joint for transfemoral amputees. Methods In this study, the gait experiment of transfemoral amputees was performed under different alignment conditions, the data of lower limb motion and ground reaction force (GRF) were analyzed and compared with those of the non-amputees. A three-dimensional finite element model of knee joint was build and used to analyze the effect of different loading modes on the internal contact mechanical characteristics between femur cartilage, tibia cartilage and meniscus. Results For the knee joint of the non-amputees, the contact force was mainly concentrated on the medial sides at the moment of the first GRF peak, while the contact force was mainly concentrated on the lateral sides at the moment of the second GRF peak. However, for the intact knee joint of the transfemoral amputees, the contact force was mainly concentrated on the medial side during the gait cycle. The Mises stress of the medial meniscus, the contact force and contact area between the medial meniscus and cartilage were all obviously increased under the alignment of 6° socket adduction. Conclusions To some extent, this study explained what changes occur in the internal contact of the intact knee due to amputation or abnormal alignment, and how these changes might be related to the development of knee OA. Then, based on the biomechanical analysis, some suggestions about prosthetic alignment were proposed.