Addressing the problem of a single activated sludge microscopic image under high magnification of a small field of view and limited information for characterizing sludge samples, a multi-image stitching approach of activated sludge microscopic images based on the Floyd algorithm is proposed. First, the scale-invariant feature transform algorithm is used to extract feature points of activated sludge microscopic images, and the distance matrix of multi-image feature matching points is computed using cosine distance. Next, the Floyd algorithm is used to determine the multi-image stitching reference map and the optimized stitching path. Finally, the images are stitched based on the stitching path and the reference map by an affine transformation. The experimental findings show the efficiency of the approach proposed in this study. In the case of a limited field of microscopic and multiple images in disorder, the approach can solve the problem of stitching images.