We developed a paper-based analytical device (μPAD) combined with self-signaling polydiacetylene (PDA) liposomes for convenient visual neomycin detection. The simple dot array type of μPAD was fabricated by the wax printing technique, and the PDA liposomes in the aqueous solution were facilely immobilized onto the hydrophilic dot region of the paper substrate. We found that, when the PDA liposomes were inserted to the paper matrix, the stability of the PDA liposomes can be significantly enhanced by adding a hydrophilic reagent such as polyvinyl alcohol and glycerol to the liposome solution. In particular, polyvinyl alcohol (PVA) provides the best stabilization among the various hydrophilic reagents tested in this contribution, and the enhanced stability sharply increased the sensitivity of the PDA liposomes in the paper matrix. Based on the above results, we successfully detected neomycin through both naked-eye observation and fluorescence measurement of PDA signals. The detection limit was 1 ppm and was selective to non-aminoglycoside antibiotics.