This paper introduces a method for measuring 3D tibiofemoral kinematics using a multi-channel A-mode ultrasound system under dynamic conditions. The proposed system consists of a multi-channel A-mode ultrasound system integrated with a conventional motion capture system (i.e., optical tracking system). This approach allows for the non-invasive and non-radiative quantification of the tibiofemoral joint’s six degrees of freedom (DOF). We demonstrated the feasibility and accuracy of this method in the cadaveric experiment. The knee joint’s motions were mimicked by manually manipulating the leg through multiple motion cycles from flexion to extension. To measure it, six custom ultrasound holders, equipped with a total of 30 A-mode ultrasound transducers and 18 optical markers, were mounted on various anatomical regions of the lower extremity of the specimen. During experiments, 3D-tracked intra-cortical bone pins were inserted into the femur and tibia to measure the ground truth of tibiofemoral kinematics. The results were compared with the tibiofemoral kinematics derived from the proposed ultrasound system. The results showed an average rotational error of 1.51 ± 1.13° and a translational error of 3.14 ± 1.72 mm for the ultrasound-derived kinematics, compared to the ground truth. In conclusion, this multi-channel A-mode ultrasound system demonstrated a great potential of effectively measuring tibiofemoral kinematics during dynamic motions. Its improved accuracy, nature of non-invasiveness, and lack of radiation exposure make this method a promising alternative to incorporate into gait analysis and prosthetic kinematic measurements later.