This study proposes a multiplane activated sludge microscopic image fusion algorithm based on discrete cosine transform (DCT) to construct a clear and large-depth-of-field microscopic view of activated sludge microorganisms. First, the edge sharpening is pretreated for the microimages with the same position and different focal planes, and the transformation coefficients of the segmented images are calculated respectively. Second, the sub-block with a large coefficient variance is selected as the sub-block of the fused image, and inverse DCT is employed to perform image fusion. Finally, the two-sided filter consistency check is used to further modify the fused image. The microimages of activated sludge collected from a sewage treatment plant are studied by image fusion experiment. Experimental results reveal that the proposed algorithm has advantages in terms of image fusion clarity and computational complexity.