Histologic methods are destructive and provide only two-dimensional images, whereas three-dimensional information is difficult to obtain. Simple and noninvasive techniques to make up for the shortcomings of histologic experiments are needed. In this study, we investigated the use of micro-CT with a contrast agent for the characterization of fixed mouse spinal cords as a means of assessing micro-structures ex vivo. In addition, we tested the possibility of using contrast agent concentrations for quantitative assessment of ex vivo micro-CT imaging. The spinal cords were soaked in nonionic iodinated contrast agents, and three-dimensional micro-CT was performed. Soaking of the mouse spinal cords in contrast agent resulted in clear differences in signal between the gray matter and the white matter at three planes, and well-defined micro-structures of nerve and bone were observed with the use of three-dimensional micro-CT data. We confirmed the potential of ex vivo micro-CT with contrast agent for quantitative assessment of the concentrations.