Background: Currently the clinical standard for measuring the motion of the bones in knee joints with sufficient precision involves implanting tantalum beads into the bones. These beads appear as high intensity features in radiographs and can be used for precise kinematic measurements. This procedure imposes a strong coupling between accuracy and invasiveness. In this paper, a tri-plane B-mode ultrasound (US) based non-invasive approach is proposed for use in kinematic analysis of knee joints in 3D space.Methods: The 3D analysis is performed using image processing procedures on the 2D US slices. The novelty of the proposed procedure and its applicability to the unconstrained 3D kinematic analysis of knee joints is outlined. An error analysis for establishing the method's feasibility is included for different artificial compositions of a knee joint phantom. Some in-vivo and in-vitro scans are presented to demonstrate that US scans reveal enough anatomical details, which further supports the experimental setup used using knee bone phantoms.Results: The error between the displacements measured by the registration of the US image slices and the true displacements of the respective slices measured using the precision mechanical stages on the experimental apparatus is evaluated for translation and rotation in two simulated environments. The mean and standard deviation of errors are shown in tabular form. This method provides an average measurement precision of less than 0.1 mm and 0.1 degrees, respectively.Conclusion: In this paper, we have presented a novel non-invasive approach to measuring the motion of the bones in a knee using tri-plane B-mode ultrasound and image registration. In our study, the image registration method determines the position of bony landmarks relative to a B-mode ultrasound sensor array with sub-pixel accuracy. The advantages of our proposed system over previous techniques are that it is non-invasive, does not require the use of ionizing radiation and can be used conveniently if miniaturized.