The integration of a star tracker and gyroscope units (GUs) can take full advantage of the benefits of each, and provide continuous and accurate attitude information with a high update rate. The systematic error calibration of the integrated system is a crucial step to guarantee its attitude accuracy. In this paper, a comprehensive calibration method for the star tracker and GUs integrated system is proposed from a global perspective. Firstly, the observation model of the predicted star centroid error (PSCE) with respect to the systematic errors including the star tracker intrinsic parameter errors, GUs errors and fixed angle errors is accurately established. Then, the systematic errors are modeled by a series of differential equations, based on which the state-space model is established. Finally, the systematic errors are decoupled and estimated by a Kalman filter according to the established state-space model and observation model. The coupling between the errors of the principal point and subcomponents of the fixed angles (i.e., normalΨx and normalΨy) is analysed. Both simulations and experiments indicate that the proposed method is effective at estimating the systematic errors of the star tracker and GUs integrated system with high accuracy and robustness with respect to different star centroid accuracies and gyroscope noise levels.