A micropatterned polydiacetylene (PDA) chip, utilizing the unique fluorogenic property of PDA and a specific biotin-streptavidin (STA) interaction, is constructed to detect pathogen infections. To construct the PDA chip, biotin-modified diacetylene liposomes are immobilized on aldehyde glass and conjugated with STA, followed by UV irradiation to polymerize the STA-functionalized diacetylene liposomes. Genomic DNA of a model pathogen, Chlamydia trachomatis, is isolated from human samples and biotin-labeled target DNA is obtained through PCR amplification using biotin-11-dUTP. Owing to the stimulus caused by the biotin-STA interaction, the biotinylated DNA induces an intense fluorescence signal on the immobilized PDA. By using this strategy, it is possible to diagnose Chlamydia infections by applying DNA samples from several nonhealthy humans to a single PDA chip. The results of this study serve as the basis for a new strategy for fluorogenic PDA microarray-based diagnosis of pathogen infections.