A self-calibration method utilizing spherical constraints is proposed for calibration of robot end-effectors. The method establishes a mathematical model to account for both the geometric errors of the robot and the deformation errors of the end-effector. A nonlinear least-squares parameter identification technique based on spherical constraints is employed to achieve autonomous calibration of the end-effector. Contrasted with methodologies relying on point plane or distance constraints, this novel technique delivers superior positioning accuracy, streamlined operational procedures and enhanced efficiency. Both simulation and experimental validation confirm that the self-calibration method using spherical constraints improves the positioning accuracy of the robot end-effector from 3 mm to 0.3 mm, showing the effectiveness of the method.