Pixel non-uniformity is an important index in the evaluation of CCD imaging performance, which reflects the difference of pixel structure itself. The traditional method of calculating DSNU and PRNU based on gray value fails to consider the temporal noise introduced by the readout circuit, and the error caused by different pixel′s different exposure time isn′t excluded, the calculation results were also only applicable to a specific exposure condition. Based on the analysis of CCD signal flow, the influence factors of gray value non-uniformity were clarified. Referring to the calculation method of DSNU and PRNU, combined with the working mode of frame-transfer CCD, a new method which calculated the dark current non-uniformity DCNU and the photo current non-uniformity PCNU was put forward. The method including setting multiple exposure time, and the multi-frame dark (bright) images was collected under each exposure time, then fitting for the dark current ("dark current+photo current"). Meanwhile, a CCD pixel non-uniformity test system was established, the stability and uniformity of the light source were verified to exclude the temporal and spatial error introduced by the test system when collecting images. On the basis of the test system, the DCNU 25.51(e-/pixel·s-1), PCNU 0.98% of CCD were measured using the new method. The result more accurately reflects the non-uniformity of CCD pixel structure, and was more universally applicable, compared to traditional DSNU and PRNU value.