Aiming at the practical requirements of accurate alignment between high-precision star sensor and the test equipment in ground calibration experiments, a nanoscale five-dimensional adjustment frame for calibration of star sensors is designed. The influence of optical axis alignment error on star sensor test was analyzed in detail. In order to ensure docking accuracy, the design idea of adopting the mechanical structure of stacked type was put forward. Then, the design scheme of a two-dimensional translation module and the three-dimensional adjustment module were discussed. After that, the simulation modeling and mechanical analysis of the adjustment frame were carried out. Finally, verification experiments were conducted on the designed micro-adjustment mechanism. The testing results show that, the displacement resolution can reach 25 nm, the angle resolution can reach 0.1 arcsecond; and the stability is quite high. Therefore, the designed micro-adjustment mechanism can meet the technical indicator requirements of accurate docking between star sensor and test equipment, and it can ensure calibration credibility of the star sensor.