Hybrid thin biomacromolecular films were fabricated by combining the in situ formation of silver nanoparticles (Ag NPs) in gelatin and self-assembly of carboxymethyl chitosan (CCHI). The hybrid films exhibited excellent antibacterial activity toward Escherichia Coli. When the fluorescent dye 4,7-(9,9ʹ-bis(6-adenine hexyl)fluorenyl)-2,1,3-benzothiadiazole modified with nucleotide adenine and quaternary ammonium groups (OFBT-AA) was adsorbed onto the surface of the hybrid films, metal-enhanced fluorescence resulted in a maximum 5.3-fold increase in the fluorescence of OFBT-AA.