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Layer-by-layer (LbL) self-assembly is one of the unique methods for fabrication of ultra-thin film. This technique has immense application aross many fields in today’s life. In this paper, we have presented the fabrication of the thin film comprising of antifungal and antibacterial cationic dye methylene blue with negatively charged E. coli K30 bacterial polysaccharide sharing with PAH. The multilayers were characterized by UV–Vis absorption spectra and AFM. Various photophysical behaviors were investigated. Such studies provide a new light in the research of layer by layer self-assembly technique.