Edible antibacterial three-layer films based on gelatin–chitosan/nisin–corn starch (GEL-CS/N-COS) were developed by layer-by-layer method. The microstructure and intermolecular interaction of the three-layer film was analyzed by SEM, ATR/FTIR, and XRD. Layer-by-layer technology enhanced the ultraviolet–visible light barrier property and elongation at break. In addition, the water solubility was reduced. Nisin was added into the three-layer film to further enhance its thermal stability and antibacterial activity. Importantly, GEL–CS–COS system exhibited good controlled release behavior of nisin. Cherry tomatoes were stored in GEL-CS/N-COS film packaging, and the effect on the shelf life of cherry tomatoes was evaluated. The results show that weight loss and total bacterial count were reduced, and higher firmness and better color were observed during storage for 22 days. These results suggest that developed three-layer film loaded with nisin is a potential antibacterial and edible material for food packaging applications.