Objective To make three dimensional(3D) reconstruction of corticospinal tracts in rat, using serial sections of rat’s spinal cord and tool software for three dimensional reconstruction. Methods The serial transversal sections of rat’s spinal cord were made and mounted directly, then stained with Luxol fast blue(LFB) and their images were taken. The section images were registered, segmented and changed into grayscale images under the Photoshop circumstance. 3D-DOCTOR software was used to make threshold segmentation and to reconstruct the spinal cord, grey matter and bilateral corticospinal tracts of rat. The reconstructed structures were observed at random angles, dissected and measured on a common desktop. Results The reconstructed spinal cord, grey matter, bilateral corticospinal tracts of rat were solid. In C1 to S4 segments of rat’s spinal cord, the corticospinal tracts lay in the posterior funiculus and were close to the back of intermediate zone, appearing as gradually narrowing columns. Conclusion The corticospinal tracts in rat’s spinal cord can be reconstructed and offer survey data by using serial LFB stained spinal cord sections and computer