The locomotor networks in the spinal cord coordinate muscle contractions and govern limb movements, ensuring postural body stability and smooth locomotion at different speeds. Dissecting the organization and connection of spinal motor networks is crucial to interpret their functional roles and thus can help to design more specific interventions for motor system diseases. Traditional two-dimensional (2D) histological sectioning is inadequate to accurately dissect complex architecture of the spinal cord since it provides only partial spatial information about spinal neural circuits. It is particularly difficult for quantitative analysis of axon regeneration after injury with 2D tissue sections, because it shows axonal fragments rather than spatial trajectory of regenerating axons. Therefore, three-dimensional (3D) imaging and analysis are extremely necessary for investigations of spinal cord structure and function. Although 3D spatial structure of the spinal cord can be reconstructed by serial sectioning, this approach is laborious and prone to image distortion. The recently emerging tissue clearing technique enables 3D imaging of the entire spinal cord at cellular resolution without tissue sectioning. The development of tissue clearing contributes to revealing the organization and function of spinal circuits and elucidating associated mechanisms underlying certain behaviors in health and disease. In this paper, we give an overview of the current clearing methods and introduce available labeling and imaging techniques as well as data processing software. Finally, we demonstrate the recent applications of tissue clearing in the spinal cord. Tissue clearing technique provides a novel tool for 3D imaging and quantification of the spinal cord, and benefits investigations of structure-function relationship of spinal networks. This review might help researchers to find the potential of tissue clearing in the studies of spinal cord and select appropriate clearing protocol for their experimental schemes.This article is protected by copyright. All rights reserved.