To elucidate the proteins associated with cochlear development and auditory formation from a histomorphological point of view, this study examined the spatio-temporal expression pattern of nestin, parvalbumin, and otoferlin in the mouse cochlea from embryonic day 17 (E17) to postnatal day 28 (P28) using immunofluorescence. Our findings revealed that nestin was broadly expressed in developing otic mesenchyme cells beneath the basilar membrane, medial to the greater epithelial ridge, and adjacent to the developing stria vascularis during late embryonic stages (E17 and E18.5). From P1 to the onset of hearing (P14), nestin was primarily expressed in fibrocytes derived from otic mesenchyme cells in the spiral ligament and spiral limbus, as well as in tympanic border cells. Dual immunofluorescence staining of nestin with Isolectin B4 (IB4), a specific vascular endothelial marker, showed the location of nestin in the blood vessels within the cochlear lateral wall. Notably, in adults (P28), nestin expression was downregulated in the fibrocytes of the spiral ligament and spiral limbus but persisted in the tympanic border cells. Parvalbumin immunolabeling was consistently observed in spiral ganglion neurons (SGNs) and inner hair cells (IHCs) from E17 through adulthood. By P1, parvalbumin expression extended to all three rows of outer hair cells (OHCs) and persisted into adulthood. Transient parvalbumin expression was also noted in afferent nerve fibers innervating the IHCs during early postnatal stages. Otoferlin labeling was predominantly detected in the cytoplasm of IHCs, with limited temporal expression in OHCs from P6 to P10. Taken together, these results illustrated the dynamic expression of nestin, parvalbumin and otoferlin during cochlear development and suggested their important function in cochlear development.