A background suppression method based on B-spline surface fitting was proposed to solve the problem of the accuracy of Mura detection, which is often caused by the uneven brightness and complex background when employing machine vision on Mura detection of TFT-LCD. Under the constraint of least square criterion, bicubic B-spline surface fitting algorithm was deployed to fit the uneven brightness background and a fairing item was added to adjust fitting precision. In order to increase the performance of the proposed algorithm and decrease the calculation time, divided fitting method was used on original image and the bicubic B-spline function was analyzed into one-dimensional functions for efficiently solving. Furthermore, pathological problems caused by solving of the two-dimensional functions could be avoided simultaneously. The effect on the Mura defect segmentation caused by uneven brightness background was eliminated by subtracting the fitted background from the original image. The experimental results show that the proposed method can suppress the uneven brightness background efficiently and Mura defect can be segmented accurately after background suppression.