The structure deformation of a micro-spacecraft in orbit caused by launching vibration and its thermal deformation resulted from sunlight will change the optical focal length of an Inertial Stellar Compass ( ISC) in the calibration before launching. Furthermore, it will affect on the high-precision attitude determination for the micro-spacecraft. To improve the calibration, an online fast calibration method based on the attitude matrix criterion was presented. Firstly, this method was used to analyze the mapping relation between the optical focal length and the attitude matrix. Then, combining the attitude matrix generated by the filter in any time with the orthogonal unit features of attitude matrix, the focal length of ISC was calibrated online based on an iterative method. Experimental results indicate that the calibration precision by proposed method for the optical length of the ISC system is equivalent to the extracting precision by the stellar center of mass method, and it is about 0.01 pixel. This method can calibrate the focal length at any time in orbit, and have fast calibration speeds and better calibration results without capturing a large number of other attitude measurement data from sensors.