A microarray-chip assay system for the fluorescence detection of phosphate-containing analytes in aqueous media has been constructed from stimuli-responsive polymerized poly(diacetylene)-liposomes for the first time. Proper combination of the liposome components (Zn(II)-dipicolylamine for phosphate binding and an amine-terminated component for anchoring the liposome onto an aldehyde-derivatized glass plate), has led to a microarray chip that selectively detects pyrophosphate, an important biomarker, over competing anions, such as phosphate and adenosine triphosphate, with nanomolar sensitivity. The chip-based assay shows advantages, such as high specificity and sensitivity, over solution-based assays that use the same liposomes, and over known homogeneous molecular sensing systems.