Polydiacetylene supramolecules (PDAs) are unique sensing materials. Upon environmental stimulation, blue PDAs can undergo a colorimetric transition from blue to red accompanied by fluorescence enhancement. In this paper, we report a new PDA system polymerized from a mixed liposome comprising 2-(2-(2-hydroxyethoxy)ethoxy)ethyl pentacosa-10,12-diynoate and pentacosa-10,12-diynoic acid at a 3:7 ratio. The PDA system provided new colorimetric and fluorometric assay methods for screening acetylcholinesterase and its inhibitors through three processes. First, myristoylcholine reacted with PDAs, which then underwent colorimetric and fluorometric transition. Second, acetylcholinesterase catalyzed the hydrolysis of myristoylcholine into tetradecanoic acid, which reduced the myristoylcholine concentration and led to faded color and fluorescence. Third and last, acetylcholinesterase inhibitors retarded the activity of acetylcholinesterase, thereby inducing the recovery of color and fluorescence.