As an efficient method of information fusion, multi-focus image fusion has attracted increasing interests in image processing and computer vision. A multi-focus image fusion algorithm based on discrete Walsh-Hadamard transform (DWHT) and guided filtering is proposed. First, a new focus region detection method is proposed, which uses DWHT and calculates L1 norm to obtain the initial decision map; then, the mathematical morphology method and guided filtering optimization are used to generate the final decision map; finally, the fused image is obtained by using the pixel-wise weighted-averaging rule and the final decision map. In order to verify the effectiveness of the proposed algorithm, three groups of multi-focus images commonly used in research are selected for experiments, and the proposed algorithm is applied to two groups of multi-focus sequence images collected in practical application, compared with other algorithms, the proposed algorithm shows obvious advantages in subjective qualitative analysis and objective quantitative evaluation indicators. Experimental results show that compared with other multi-focus image fusion algorithms, the proposed algorithm can extract the focus region from the source image more effectively, and enhance the detail retention ability and spatial continuity of the fused image.