Five-axis 3D printing equipment enables the printing of complex curved surfaces with varying curvatures, playing a significant role in the aerospace industry. Calibration is a crucial step in ensuring printing accuracy of such equipment. To achieve rapid calibration of five-axis 3D printing equipment and improve its printing precision, a calibration method utilizing a calibration object is proposed. Based on screw theory, a transformation model between the machine tool coordinate system and workpiece coordinate system of the five-axis 3D printing equipment is established, and the quantities to be calibrated are determined. A calibration object containing five calibration spheres is designed, and coordinates of the calibration spheres’ centers in the machine tool coordinate system are obtained using the least squares fitting method for sphere centers. An equation for calibrating the linear axes is established based on transformation relationship between the calibration coordinate system and machine tool coordinate system, while the rotational axes are calibrated using plane normal vector solving and the least squares fitting method for circles. 3D printing experiments are conducted based on the calibration results afterwards. Point cloud fitting analysis reveals that the average deviation of the printed samples by the five-axis 3D printing equipment after calibration reduced by 91.3% compared with before, significantly enhancing printing accuracy.