The development of mirror-image biology systems faces a crucial barrier of lacking an L-DNA sequencing technique. Here, we developed a practical method for sequencing mirror-image DNA by adopting the Maxam-Gilbert sequencing approach, through which specific nucleobases in an end-labeled L-DNA are cleaved by achiral chemicals. This technique may facilitate the therapeutic application of nuclease-resistant L-aptamer drugs, and bring the vision of building an alternative, mirror-image self-replicating system closer to reality.