Intraepidermal nerve fiber (IENF) density is commonly evaluated to diagnose peripheral neuropathy. However, conventional two-dimensional (2D) analysis using rodent models shows high interstudy variability. Three-dimensional (3D) IENF analysis has been proposed for human skin biopsies because the spatial location of each nerve can be easily determined. However, no studies have compared 2D and 3D analyses of mouse cutaneous nerve fibers under the same conditions. We aimed to establish a more accurate analysis method for mouse cutaneous nerve fibers. We used the glabrous plantar metatarsal skin of male C57BL/6J mice. The middle area of the plantar skin was used for 2D and 3D analyses, and the marginal area was also investigated in the 3D analysis. Tissue transparency, nerve fiber-specific antibodies, confocal microscopy, and IMARIS software were used for the 3D analysis. The 3D analysis clearly defined branching points and continuity, allowing accurate IENF density measurement. Conversely, the 2D analysis could not accurately determine IENF density because it could not detect the continuity of the nerve from the dermis to epidermis. Thus, the actual IENF density from the 3D analysis was significantly less than that from the 2D analysis. In addition, the density and length of IENFs in the middle area were significantly higher than those in the marginal area. This 3D approach enables the precise capture of IENF trajectories with various parameters, establishing a standard method for evaluating peripheral neuropathy models. Furthermore, our findings indicate that comparative studies aiming to analyze mouse IENF need to consider the site of skin sampling.