In order to estimate the sensitivity limitation of the polydiacetylene (PDA) sensor system, the response of the PDA system to several sulfonate salts and amines were examined. The PDA systems were fabricated with N-(2-aminoethyl)pentacosa-10,12-diynamide (AEPCDA) and 10,12-pentacosadiynoic acid (PCDA). The functional group at the end of the PDA system is crucial to sensing the substrate because this functional group interacts with the substrate. The sensitivity limitation toward the molecular weight and the concentration of the substrate is also revealed. The pKa of the amines is less crucial to the sensing than the molecular weight of the substrate.