The increasing number of analytes in concern and the alarming health and environmental consequences have required effective means of monitoring for safety control. Biosensors offer advantages as alternatives to conventional analytical methods because of their inherent specificity, simplicity, and quick response. Colorimetric biosensor, one of biosensor group, is one of the easiest and the most convenient methods because detection can be done using naked eye. Recently, a novel method for rapid detection and read-out of specific immunoassays with naked eye using polydiacetylene (PDA) was developed. Polydiacetylene has recently been in the limelight as a transducing materials because of its special features that allow optical transduction of sensory signals and inherent simplicity and ease of use in supramolecular chemistry. Various forms of PDA are used as a sensor platform for detection of various biological analytes such as viruses, DNA, proteins, bacteria and hazardous molecules.