Greenhouse gases monitoring instrument (GMI) uses spatial heterodyne interference technology which can effectively detect atmospheric high-resolution absorption spectra during 759～2058 nm. And the calibration is the base of GMI spectral image data quantitative application. Based on the principle of GMI imaging and spectral calibration, the methods of on-orbit spectral calibration are discussed, and spectral calibration scheme of the external light source characteristic spectral is presented. Through the calculation of the simulation data, the uncertainty of calibration is further analyzed, and the uncertainty of on-orbit the spectral calibration is 0.030 nm. The calibration results show that the calibration uncertainty is mainly affected by the light source calibration uncertainty and regression uncertainty. The method meets the calibration requirements of the instrument and provides the basis for quantitative inversion of atmospheric main greenhouse gases.