Tracking joint motion of the lower extremity is important for human motion analysis. In this study, we present a novel ultrasound-based motion tracking system for measuring three-dimensional (3D) position and orientation of the femur and tibia in 3D space and quantifying tibiofemoral kinematics under dynamic conditions. As ultrasound is capable of detecting underlying bone surface noninvasively through multiple layers of soft tissues, an integration of multiple A-mode ultrasound transducers with a conventional motion tracking system provides a new approach to track the motion of bone segments during dynamic conditions. To demonstrate the technical and clinical feasibilities of this concept, an in vivo experiment was conducted. For this purpose the kinematics of healthy individuals were determined in treadmill walking conditions and stair descending tasks. The results clearly demonstrated the potential of tracking skeletal motion of the lower extremity and measuring six-degrees-of-freedom (6-DOF) tibiofemoral kinematics and related kinematic alterations caused by a variety of gait parameters. It was concluded that this prototyping system has great potential to measure human kinematics in an ambulant, non-radiative, and noninvasive manner.