Cartilage-bone interface (CBI) is a complex structure which bears important information in pathophysiology of osteoarthritis (OA). While high-frequency ultrasound (US) has been widely used for the investigation of articular cartilage, 3D imaging of CBI using US is less commonly reported in this field. Here, we adopted a 3D high-frequency ultrasound imaging approach specifically for the investigation of CBI in human knee samples. Fifteen osteochondral disks from the tibial plateau of seven OA patients were prepared in vitro and scanned using both high-frequency US and micro-CT imaging. The 3D morphology of the tidemark was reconstructed and compared using an image registration approach between the two imaging modalities. Results showed that the 3D tidemark could be well registered between the two imaging methods with a mean surface discrepancy of 33.2 ± 9.9 μ m . Quantitative surface waviness/roughness parameter analysis showed significant correlations between the two imaging modalities. An intensity projected en face imaging was proposed to probe characteristic details of the CBI such as its perforations. This study provided evidence for the 3D high-frequency ultrasound as a nonionizing radiation imaging tool potentially useful to evaluate the change of CBI in basic research of join diseases including OA.