The spinal cord connects the brain to the peripheral nervous systemand plays an important role in encoding downstream brain commands and peripheral input signals during somatosensory and motor processes. Resolving the relationship between animal behavior and spinal cord neural activity through optical imaging remains a major challenge in neuroscience research. We have developed a method for in vivo imaging of the spinal cord in freely behaving mice using miniaturized two-photon fluorescence microscopy. The image quality and imaging stability of the method were evaluated by imaging the anterior spinal artery vessels, during spontaneous behavior and strenuous movements of freely behaving animals in an unrestrained environment. This physically solves the key problems in previous studies, such as image bias and loss due to the irregular motion of the spinal cord, and it achieves stable imaging of the spinal cordinfreely behaving mice based on miniaturized two-photon fluorescence microscopy. In addition, this method enables real-time imaging of individual neuronal calcium signals in the superficial neuronal activity of the spinal dorsal horn. This will provide technical support to explore the neuronal activity patterns in the spinal cord during somatosensory and motor processes, it’s important for advancing the study of spinal cordrelated functional neural networks.