Space-borne differential optical absorption spectrometer is a nadir viewing wide field imaging spectrometer, which adopts two-dimensional CCD detector arrays. The pixel response non-uniformity exists in each column of spatial dimension, which will introduce high-frequency instrument-related spectral structures in the measurement data. However, the non-uniformity calibration of space-born imaging spectrometer is difficulty due to two factors: the spectral smile effect and the large field of view. For this reason, a method of non-uniformity calibration is discussed in detail. The result shows that the spectral smile effect and non-uniformity of full FOV image are corrected effectively, and high-frequency instrument-related structures in the measurement data are removed.