Collagen was used to construct corneal repair materials because of their good physicochemical qualities, excellent biocompatibility, and low immunogenicity. However, the issue of bacterial infection during application process limited their clinical applications. To solve this problem, a novel form of antibacterial collagen-based film for corneal repair was suggested in this study. This membrane took type I collagen as raw material and used 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) as cross- linking agents to effectively incorporate aminoglycoside gentamicin molecules onto the surface of the collagen film. The structure of the gentamicin/collagen (GM/Col) film was characterized by infrared spectrum and scanning electron microscopy (SEM). At the same time, the drug loading of gentamicin on the GM/Col film was studied, and the antibacterial performance of the modified film was evaluated by Staphylococcus aureus and Escherichia coli. The results show that the mass of gentamicin loaded on GM/Col membrane accounts for about 2.33%±0.54% of its total mass. The successful incorporation of gentamicin significantly improves the antibacterial properties of the collagen membrane. In addition, the GM/Col film has good physicochemical qualities and excellent biocompatibility. As a result, there are numerous possible uses of this GM/Col film in corneal tissue engineering.