Antibacterial materials are of great importance in preventing bacterial adhesion and reproduction in daily life. Silver nanoparticle (AgNP) is a broad-spectrum antibacterial nanomaterial that has attracted significant attentions for its ability to endow natural materials with antibacterial ability. Silk sericin (SS) has a great advantage for biomaterial application, as it is a natural protein with excellent hydrophilicity and biodegradability. In this study, we prepared AgNPs and polyelectrolyte membrane (PEM) modified SS/Agar films through the layer-by-layer adsorption technique and ultraviolet-assisted AgNPs synthesis method. The film was well characterized by scanning electron microscopy, energy dispersive spectroscopy, X-ray diffraction, fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy. Other properties such as water contact angle, wettability and tensile strength, the release of silver were also studied. The antimicrobial activity of AgNPs-PEM-SS/Agar film was investigated against Escherichia coli and Staphylococcus aureus as the model microorganisms by the inhibition zone and bacterial growth curve assays. The results suggested that the AgNPs-PEM-SS/Agar film had excellent mechanical performance, high hydrophilicity, prominent water absorption ability, as well as outstanding and durable antibacterial activity. Therefore, the prepared novel AgNPs-PEM-SS/Agar composite film is proposed as a potentially favorable antibacterial biomaterial for biomedical applications.