〔Abstract〕 Objective To investigate how vitamin B12 eye drops regulate MMP-9 and TNF-α expression levels in the corneas of dry eye disease（DED）mouse model induced by a combination of an artificially simulated dry environment（air humidity <30%）and subcutaneous injection of hyoscine hydrobromide. Methods Fifty C57BL/6 female mice were used in the study. 10 mice formed A group（blank control），while the remaining 40 mice were subjected to dry eye disease induction through subcutaneous injection of scopolamine hydrobromide in a controlled environment with <30% air humidity. All mice underwent schirmer I test（SIT），tear break-up time（TBUT）， and corneal fluorescence staining score（CFS）evaluations. Mice meeting modeling criteria were randomly assigned to three groups：B group（model group），C group（PBS control），and D group（vitamin B12 group）. 0，7，14，and 21 days after treatment，each group received TBUT，SIT，CFS，and corneal inflammation assessments. Macroscopic analysis evaluated the efficacy of vitamin B 12 eye drops in treating dry eye. After 21 days of treatment，all mice were euthanized via cervical dislocation and their corneas analyzed using HE staining to examine morphologi ⁃ cal changes. ELISA and immunohistochemical methods were employed to detect MMP-9 and TNF-α expression lev⁃ els in corneal tissues across groups. Results In dry environments（air humidity <30%），subcutaneous injection of scopolamine hydrobromide induced changes in dry eye-related indicators in mice：SIT significantly decreased， TBUT significantly shortened ，corneal fluorescein staining increased ，and CFS all significantly elevated（P < 0. 05），indicating successful establishment of the dry eye model. A total of 30 dry eye mice were enrolled. When the dry eye model was successfully established without drug intervention，no statistically significant differences were observed between B group，C group and D group in terms of TBUT，SIT，corneal fluorescein staining，or CFS. Compared with A group，B group，C group and D group showed statistically significant differences in re ⁃ duced SIT，shortened TBUT，increased corneal fluorescein staining，and elevated CFS（P <0. 05）. After drug intervention，D group（vitamin B12 group）exhibited significantly increased SIT，prolonged TBUT，reduced corneal fluorescein staining，clearer corneas，decreased fluorescein staining，and lower CFS compared with B and C groups （P<0. 05）. ELISA and immunohistochemical staining results showed that compared with A group，B and C groups exhibited significantly increased expression of MMP-9 and TNF-α in corneal tissues（P <0. 05）. D group showed decreased expression of MMP-9 and TNF-α compared with B and C groups but remained higher than A group（P < 0. 05）. Conclusion The expression of MMP-9 and TNF-α in the corneal tissues of mice with dry eye is elevated， suggesting that both inflammatory factors，MMP-9 and TNF-α , are involved in the development of dry eye. Vitamin B12 eye drops may improve the symptoms of ocular surface discomfort by promoting the repair of the corneal epithelium. Vitamin B12 eye drops may increase tear secretion，prolong tear film break up time，reduce corneal fluorescein staining，and alleviate the inflammatory reaction of the cornea by decreasing the expression of MMP-9 and TNF-α in the corneal tissues of the mice，thus playing a therapeutic role in the treatment of dry eye disease.