Objective To quantitatively analyze the in-vivo kinematics of the tibiofemoral joint in patients with arthrofibrosis after anterior cruciate ligament reconstruction ( ACLR) surgery. Methods Fifteen patients with arthrofibrosis after ACLR and 15 healthy subjects were included in the study. The tibiofemoral motion trajectories relative to the femur during weightbearing and non-weightbearing knee flexion of the subjects were collected by using a portable three-dimensional (3D) knee motion analysis system, and kinematic data of the tibiofemoral joint with 6 degrees of freedom (6 DOF) were obtained. Results Compared with healthy subjects, the internal tibiarotation was significantly decreased in patients with arthrofibrosis under 30°, 45°, 60° and 75° weightbearing knee flexion (P<0. 001), and the tibial valgus was significantly decreased in patients with arthrofibrosis under 30° and 45° weightbearing knee flexion (P<0. 05). The internal tibial displacement in patients with arthrofibrosis was significantly smaller than that in healthy subjects under 75° non-weightbearing knee flexion (P<0. 05). Conclusions Knee arthrofibrosis limits the rotation and internal displacement of the tibia, but has little impact on the anterior and posterior displacement of the tibia relative to the femur. Therefore, in clinical practice, various treatments should be used to release the adhesion in the medial and lateral femoral grooves and the contracturof the lateral collateral ligaments, address the limitation in rotation and displacement movement, so as to restore knee function to the fullest extent.