Micro-arrayed polydiacetylene (PDA) vesicles mixed with phospholipids on glass slides were prepared for label-free detection of Escherichia coli. When E. coli bound to its antibodies chemically attached to polydiacetylene, the fluorescence of the vesicles was dramatically increased. The insertion of dimyristoyl phosphatidylcholine (DMPC) in the vesicles drastically reduced the response time for the fluorescence changes. Vesicles with 20-30% DMPC provided optimal results for bacterial detection. Fourier transform infrared (FTIR) spectra analysis suggested that DMPC insertion decreased the strength of hydrogen bonding among the amide and carboxylic acid groups of the polydiacetylene vesicles. Reduced bonding strength resulted in less rigid structure of the polydiacetylene polymer, allowing more rapid detection upon molecular recognition.