This study aimed to investigate the therapeutic potential of cell-free conditioned media (CM) from human mesenchymal stem cells (hMSCs), specifically stem cells from human exfoliated deciduous teeth (SHED), for treating ocular surface diseases. The proteomes of various hMSC-CMs were compared using cytokine array and liquid chromatography-mass spectrometry (LC-MS). Bioinformatic analysis identified key biological pathways associated with SHED-CM, immortalized SHED-CM (IM-SHED-CM), and a fractionated component of IM-SHED-CM in which low weight molecules (less than 3.5kD) were depleted. Corneal epithelial wound healing models were constructed by epithelial scraping and treated with eye drops derived from SHED-CM. For the migration assay, the human corneal epithelial cells were wounded and then incubated with SHEDCM. SHED-CM, IM-SHED-CM, and >3.5 kD fractionated component eyedrops were administered to a chronic graft-versus-host disease (cGVHD) mouse model with sever corneal epithelial damages. SHED-CM, IM-SHED-CM, and >3.5 kD fractionated component of IM-SHED-CM were enriched in factors involved in epithelial wound healing, particularly extracellular matrix (ECM) organization. Both in vitro and in vivo assays demonstrated that SHED-CM significantly enhanced corneal epithelial wound healing. Furthermore, SHED-CM-derived eye drops reduced corneal epithelial damage, inflammatory cell infiltration, and oxidative stress in the corneal epithelium and maintained the expression of limbal stem cell markers in the cGVHD mouse model. These findings suggest that SHED-CM eye drops could be a novel treatment for corneal epithelial damage, highlighting the role of bioactive factors in promoting wound healing and offering an alternative to cell-based MSC therapies for corneal wound healing.