Spatial calibration is a key technique in Freehand 3D Ultrasound. In this article, a two-layer Z-fiducial phantom with one “Z” shaped fiducial in each layer is used to perform spatial calibration. After freehand scanning above the phantom, the intersections of the scan plane and Z-fiducial plane in B-scans are segmented, and the calibration transformation matrix between the coordinate system of the position sensor receiver and B-scan is calculated using the least-squares fitting algorithm. At last, the calibration result is evaluated on two criteria: precision and accuracy. The result shows that our phantom is convenient as well as reliable for freehand spatial calibration.