Radiometric calibration is one of the key technologies for quantitative application of optical remote sensing satellite data. This study proposes an on-orbit radiometric calibration approach for a high-resolution optical remote sensing satellite based on sub-pixel targets. The detection reference is the reflected point source, and the ground synchronization measurement is mainly used. Atmospheric transmittance and incident pupil radiance are obtained by simplifying radiometric transfer calculation. The method can also effectively isolate the response of a remote sensing image created by sub-pixel targets from the response produced by sky path radiance, ground-atmospheric coupled radiance, and background radiance, according to multi-energy sub-pixel target setting and on-orbit point spread function detection results. The approach can break the limit of time, locale, and weather conditions and achieve high precision calibration for the optical remote sensing satellite with a full dynamic range in complex environments. Results show that calibration uncertainty for the high-resolution optical remote sensing satellite based on sub-pixel targets is lower than 3.2%. Moreover, the calibration result of the proposed method is different from that of the gray-scale target method by 3%. A small and light sub-pixel target can satisfy the high frequency on-orbit calibration application of the optical sensing satellite.