Biothiols such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) participate in numerous cellular functions and physiological processes. Because changes in the levels of these biological important substances are associated with various clinical disorders and diseases, methods for their selective sensitive and quantitative detection are in high demand. In the current study, we developed a new, selective and sensitive colorimetric and fluorometric biothiol chemosensor that is comprised of a mercury-complexed, pyridine-containing polydiacetylene (PDA). Owing to its high affinity toward sulfur, biothiols promote decomposition of the pyridine-mercury complex in the PDA in conjunction with colorimetric and fluorescence changes. Specifically, upon addition of biothiols the new chemosensor undergoes a blue-to-red color transition and generates red fluorescence. Moreover, incorporation of the chemosensor in electrospun PEO nanofibers leads to an enhanced sensitivity for biothiol detection. The strategy developed in this study should be applicable to the design of other thiol-specific sensors that utilize pyridine-containing conjugated molecules.