Objective To establish a new method for quantitative observation of inner ear hair cells, neurons and nerve fibers from the sagittal slices of temporal bones. Methods Temporal bones of 4 CBA/CaJ mice at 45 days of age were removed under deep anesthesia. Inner ear cavity in the temporal bones were perfused with 2.5% glutaraldehyde solution and immersed in the fixative for 6 hours. The temporal bones were then immersed in 2% osmium tetroxide solution for 2 hours. After decalcification, dehydration and embedding, semi-thin sections (2 μm) were cut along the sagittal plane of the temporal bones and stained with 0.5% toluidine blue. The hair cells, neurons and nerve fibers were quantitatively observed in a small field of view under an optical microscope. Results The collection and observation began with plane slices parallel to the medial wall of the temporal bone. When the medial wall of the temporal bone was cut to enter the cochlear nerve canal and the superior vestibular nerve canal, cross-sectional sections of the central end of cochlear nerve bundles and the inferior vestibular nerve bundles were obtained. When the cross-section of the macula of utricle and the crista ampullae of the superior and lateral semicircular canals is cut, the cross-section of the hair cells in above structures is obtained, and the cross-section of the cell bodies of the superior vestibular neurons and the nerve fibers at both peripheral and central ends are also obtained. When the cross-sections were cut to the macula of saccule and the crista of ampulla of the posterior semicircular canal, Cross-sectional slices of the hair cells contained therein were obtained, as well as cross-sectional slices of the cell bodies of the inferior vestibular neurons and the nerve fibers at both ends thereof. When the cross section of the bony spiral plate of the cochlea is cut, the cross section of the peripheral nerve fibers of the spiral ganglions is obtained. When the cross section of the modiolus is cut, the cross section of the cochlear hair cells and the body of spiral ganglion can be obtained. In this experiment, we found that the averaged density of hair cells in each vestibular sensory epithelium of normal mice was 18.67±1.88/180μm. The number of peripheral nerve fibers of the spiral ganglion in a single habenula perforata was 57.83±9.09. The averaged density of the spiral ganglion of the cochlea and the superior and inferior vestibular neurons was 20.39±2.23/0.0075mm2. The averaged density of the nerve fibers towards the central nervous system in the cochlear nerve, the inferior and superior vestibular nerve was 497.06±25.28/0.0075 mm2. Conclusion The observation angle of sagittal slices of the temporal bone is conducive to a comprehensive cytological quantitative evaluation of the inner ear nervous system.