Photopolymerized vesicles of 10,12-pentacosadiynoic acid (PPCDA) were successfully assembled into polyelectrolyte multilayer (PEM) thin films using either chitosan or poly(ethylenimine) (PEI) as a polycation. The PEM films assembled from chitosan or PEI polycation retained the blue color of PPCDA vesicles. For the blue films, an increase in absorbance at 635 nm as a function of the number of deposited layers of PPCDA vesicles was observed, confirming the uniform layer-by-layer deposition process. The spherical structures of PPCDA vesicles, as well as their important colorimetric responses to solvent (ethanol), pH, and temperature, are retained in the PEM film. Compared to the vesicles dispersed in water, the PEM films are much more stable to aging. Layer-by-layer assembly thus provides a convenient means to prepare colorimetric sensing devices, with extended shelf life, from polydiacetylene vesicles.