Conjugated polymers are intriguing materials that have potential practical applications in diverse interdisciplinary subjects. Among them, polydiacetylenes (PDAs) have been extensively studied due to their interesting structural, spectral, and optical features. In particular, the unique colorimetric and fluorescent transition of PDAs in response to different external stimuli makes them a novel class of sensing materials, and numerous applications of PDAs as bio- or chemosensors have been explored in the past few decades. In this review, we summarize the latest developments with regard to the applications of PDAs as a class of sensing materials presented in the literature since 2014. This review is sorted into categories based on the structural differences of diacetylene monomers, from which PDAs are generated. In addition, different forms of PDAs and various methods for improving the sensing performance of PDAs are also emphasized.