The purpose of this study was to develop a multi-layer diacetylene (PDA)-coated filter and to verify the proposed color response mechanism of PDA-coated filters to bacteria. Unlike other reports which immobilize PDA liposomes on solid support or create monolayer Langmuir films, multi-layer PDA-coated filters were generated by directly evaporating organic solvents. Different from incorporating functional headgroups or inserting phospholipids into PDA, we reported that bacterial growth can trigger the color change of PDA sensors without any modification or phospholipid insertion. The mechanism that pH change from bacteria metabolites lead to color change of PDA filter was proposed and verified by carefully designed dextrose-free medium with phenol red. Further, culturing Salmonella Typhimurium, E. coli, L. innocua and M. luteus with PDA-coated filters on phenol red agar in absence of dextrose verified the potential of applying PDA-coated filter for bacterial detection, specifically amine-producing bacteria. Thus, PDA-coated filters may be a useful tool for food safety and shelf life applications.