A new alignment calibration method of attitude sensors for the precision attitude determination of a spacecraft based on the extended Kalman filter is proposed. The proposed method is divided into two steps connected in series: the gyro and the star tracker calibration. For gyro calibration, alignment errors and scale factor errors are estimated during the calibration maneuver under the assumption of a perfect star tracker. Estimation of the alignment errors of the star trackers and compensation of the gyro calibration errors are then performed using the measurements including payload information. Performance of the proposed method are demonstrated by numerical simulations.