Objective ·To establish a standardized research platform for comprehensive imaging of the cochlea and its surrounding tissues through multi-scale resolution three-dimensional (3D) imaging, thereby enabling 3D visualization and analysis of the cochlea under physiological conditions. Methods ·The cochlea or combined cochlea and whole brain samples were obtained from 8‒12-week-old thymus cell antigen 1 (Thy1) -driven yellow fluorescent transgenic mice (Thy1-YFP-16 mice) after cardiac perfusion. Tissue clearing was performed using PEGASOS, followed by direct imaging of the cochlea and surrounding tissues through their entire depth using a laser confocal microscope with 4× and 10× objectives. For individual cochlear samples, whole tissue immunofluorescence staining was performed in combination with a neurofilament-200 (NF200) antibody and propidium iodide (PI) fluorescent dye, in the process of tissue clearing and embedding using PEGASOS and TESOS techniques. The entire cochlea was then subjected to serial sectioning with a paraffin microtome, and each section was imaged using a laser confocal microscope equipped with a 63× oil immersion objective. 3D reconstruction was performed using ImageJ for image stitching, followed by image processing, single-nerve-fiber tracking, and length measurement conducted in Imaris software. Fiber path curvature was calculated using MATLAB, and statistical analyses were performed with GraphPad Prism. Results ·Transparent cochlear and surrounding tissue samples were successfully prepared using tissue clearing techniques. The cochlea and its surrounding tissues were directly imaged by low- and medium-magnification objectives on the confocal microscope, which reconstructed the 3D morphology of the cochlea and delineated its spatial relationship with adjacent structures such as the vestibular nerve. Serial sectioning and imaging with a high-magnification objective enabled 3D imaging of the entire cochlear sample at single-cell resolution. This approach revealed the overall pathway of THY1⁺ fibers within the cochlea, extending from the hair cell region to the modiolus. Single nerve fiber tracing revealed distinct trajectory characteristics of the cochlear efferent nervous system and auditory nerve fibers near the spiral ganglion. Conclusion ·The PEGASOS and TESOS techniques enable multi-scale resolution 3D imaging of the cochlea and its surrounding tissues as an integrated whole. When combined with whole-tissue immunofluorescence staining, this methodology can be used to delineate the 3D spatial relationships between the cochlea and adjacent tissues, as well as between cells and nerve fibers within the cochlea itself. Furthermore, it allows for the tracing of individual nerve fiber pathways.