This paper presents a method for separating objects with unclear edges from the nonuniform illumination background. The paper uses an iterative lowpass filter to estimate the rough background first. And then, objects are located by removing the estimated background from the original image, meanwhile the rough segmentation is worked out too. Next, the primary segmentation output is dilated by means of the iterative lowpass filter again to connect most of the objects together as a larger one. After that, some background points are selected from the dilated image as the control points for image background reconstruction based on B-spline. Finally, the reconstructed background is removed from the original image directly to make the separation. A quantitative evaluation of the performance is also presented in this paper.