According to the actual demands of scientific CMOS (sCMOS) image sensor development project, a two-order polynomial non-uniformity correction fitting algorithm based on FPGA was designed. Firstly, calibration will produce a group of response curve of each column pixels. The least square method was used to fit the pixel response curves to the average response. The corrected coefficient will be saved in the on-board flash and correct pixel value real-time. The non-uniformity of the processed images decreases from 17.1% to 7.6%. This algorithm can effectively improve the devices’ adaptability to the characteristics of high dynamic and nonlinearity, and presents advantages of real-time calculation and correction, thus it can be easily transplanted and integrated through modular design.