Aiming to address the chicken-and-egg problem in star identification and the intrinsic parameter determination processes of on-orbit star sensors, this study proposes an on-orbit self-calibration method for star sensors that does not depend on star identification. First, the self-calibration equations of a star sensor are derived based on the invariance of the interstar angle of a star pair between image frames, without any requirements for the true value of the interstar angle of the star pair. Then, a constant constraint of the optical path from the star spot to the center of the star sensor optical system is defined to reduce the biased estimation in self-calibration. Finally, a scaled nonlinear least square method is developed to solve the self-calibration equations, thus accelerating iteration convergence. Our simulation and analysis results show that the bias of the focal length estimation in on-orbit self-calibration with a constraint is two orders of magnitude smaller than that in on-orbit self-calibration without a constraint. In addition, it is shown that convergence can be achieved in 10 iterations when the scaled nonlinear least square method is used to solve the self-calibration equations. The calibrated intrinsic parameters obtained by the proposed method can be directly used in traditional star map identification methods.