Abstract: To develop a novel, eco-friendly active food packaging material, an ε-polylysine (ε-PL)/agarose composite hydrogel film was prepared. The microstructural gelation mechanism, physicochemical barrier properties, mechanical strength, and in vitro antibacterial activity were systematically evaluated. ε-PL molecules were uniformly dispersed within the three-dimensional agarose network through intense physical entanglements. This dispersion effectively inhibited ε-PL crystallization and shifted the primary thermal degradation temperature above 300℃. The dense skeletal structure created a complex tortuous path, reducing light transmittance to 27.1% and water vapor permeability from 27.38 to 16.56 g/(m²·h). Furthermore, the tensile strength increased by 73% (reaching 9.25 MPa) while maintaining favorable spatial flexibility. In vitro assays confirmed excellent antibacterial activity against pathogens such as Escherichia coli and Staphylococcus aureus (MIC=78 μg/mL), as well as spoilage fungi including Aspergillus niger and Penicillium. Prepared via a simple process without chemical crosslinking, this composite hydrogel film integrates superior barrier and antibacterial properties, demonstrating significant potential for application in active food packaging.