Objective To reconstruct the 3D motion of the knee joint after total knee arthroplasty (TKA) and measure the kinematics and the articular contacts between the posterior stabilized TKAs. Methods Sixteen knees undergoing TKAs were scanned by fluoroscopy. An algorithm of automatic registration was developed to match the 3D TKA models and 2D images. The kinematical parameters and articular contact of the tibiofemoral joint was investigated. ResultsThe time for a single image was less than 30 seconds. The in-plane repeatability was within 0.4 mm and 0.5 degree. The application of the high-flex insert didn’t obviously improve the ability of the flexion and the internal/external tibial rotation. When the knee flexed, the contact point on the lateral side moved more posteriorly than that on the medial side. The contact between the post-cam and the femoral prosthesis occurred with the flexion at about 30 degrees. The average range of the contact was within 9 mm. Conclusions The 3D kinematics of the in vivo TKA knee joint was accurately measured by using 2D-3D automatic registration technique. The result can provide references for the biomechanical study of TKA knees and the improvement of TKA prosthesis design.