<p>Percutaneous needle biopsy is commonly performed under ultrasound guidance to visualize the target lesion and monitor needle position. This study aims to improve spatial information for the operator by accurately visualizing the ultrasound image at its true anatomical location and scale within a head-mounted display, co-registered with pre-procedural imaging data. Current mixed reality systems rely on fiducial markers to track the instruments, which limits their practical utility in clinical settings; therefore, this study evaluated a custom markerless method for ultrasound probe tracking that achieved accuracy suitable for clinical applications. By displaying the ultrasound image relative to the probe’s spatial position, the system enables seamless fusion with pre-procedural imaging data. Combined with markerless needle tracking, this approach provides a complete navigation solution. Target registration error analysis between 2D ultrasound and 3D computed tomography demonstrated a 95% likelihood that the error remains within 3.41 mm. A biopsy navigation assessment test confirmed the system’s strong potential to reduce the number of puncture attempts compared to conventional ultrasound guidance alone. By providing comprehensive spatial and imaging information, the navigation system may contribute to reduced operator error and improved procedural success rates.</p>