Utilizing the colorimetric and fluorogenic changes, a system based on polydiacetylenes (PDAs) is developed for the detection of neomycin. The PDA supramolecules polymerized from the mixed liposome composed of N‐(3‐hydroxyphenyl)pentacosa‐10,12‐diynamide (PCDA‐AP) and pentacosa‐10,12‐diynoic acid (PCDA) at an optimized ratio of 1:9 display a unique colorimetric change (blue to red) and fluorescent enhancement in the presence of neomycin. The detection limit for neomycin is estimated to be 2.55 × 10−7 M by the fluorogenic method. The optical changes induced by neomycin can be attributed to the disruption of the hydrogen bonding between phenol and carboxylic acid from PCDA‐AP and PCDA.