The detection of proteins by gel electrophoresis and subsequent staining is the most commonly used method for protein analysis, but it requires considerable time and effort as well as denatured protein samples. In this study, we developed a polydiacetylene (PDA)-based colorimetric sensor that does not require any instrument, time-consuming processes, or protein denaturation steps. Specifically, to assess the expression of glutathione S-transferase (GST)-fusion recombinant protein utilizing the interactions between the enzyme GST and its substrate, glutathione (GSH), a terminal group of PDA monomers was modified with the GSH tripeptide. The diacetylene liposome containing the GSH-modified diacetylene monomer was polymerized upon UV irradiation at 254 nm, resulting in a blue PDA liposome. The terminal GSH moiety of the PDA liposome seemed to be perpendicular to the PDA backbone, enabling its easy interaction with the active site residues of GST in solution. Using the GSH-functionalized PDA sensor, we successfully detected GST and GST-human growth hormone (GST-hGH) by the blue-to-purple chromatic transitions. As this PDA colorimetric sensor is simple, rapid, and detectable by the naked eye without protein gel electrophoresis, this strategy is of great practical value as a point-of-care testing sensor for the expression of GST-fusion protein.