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This study was designed and conducted to investigate the mechanical and physical properties of fish gelatin films and the effect of Glutaraldehyde crosslinking on antimicrobial control of poly-l-lysine. In this study, the film was prepared by casting method and then 0.05% Glutaraldehyde and 0.05% poly-l-lysine added to fish gelatin film. After that, physical and mechanical properties, antimicrobial activity and release of poly-l-lysine from the film were observed. The results showed that the addition of glutaraldehyde to the fish gelatin film increased tensile pressure (6.80 MPa) and reduced solubility (38.51%), moisture (8.05%), and water vapor permeability (2.03 mm/h mm²kpa×10^-6­). The fish gelatin film with glutaraldehyde as a crosslinking agent was showed a smooth surface without porosity according to the SEM results. Moreover, the release of poly-l-lysine from the biopolymer containing the Glutaraldehyde was slower and more continuous due to crosslinking. Considering the mechanical and physical properties of the films and release control of the antimicrobial compound, it seems that films containing crosslinking agents can be used in food storage.