This paper deals with in-flight calibration method of gyros and attitude sensors of the spacecraft on orbit. On-orbit calibration is an in-flight calibration process for more precise spacecraft attitude determination. For the spacecraft which is required more precise pointing accuracy, on-orbit calibration should be performed after the launch to accommodate attitude estimation error caused by vibration and thermal shocks. In this paper, a Quaternion based Extended Kalman Filter (EKF) is used to perform the on-orbit calibration. The observability of on-orbit calibration along trajectories is discussed by Lie-derivative-based observability analysis, and the degree of observability is computed by singular value decomposition (SVD). It is shown that the system is globally observable with calibration maneuver and the analysis results about the observable degree of each state are presented.