We describe a nanoscale strategy for detecting biotin-streptavidin binding using near-field scanning optical microscopy (NSOM) that exploits the fluorescence properties of single polydiacetylene (PDA) liposomes. NSOM is more useful to observe nanomaterials having optical properties with the help of topological information. We synthesized amine-terminated 10,12-pentacosadiynoic acid (PCDA) monomer (PCDA-NH(2)) and used this derivatized monomer to prepare PCDA liposomes. PCDA-NH(2) liposomes were immobilized on an aldehyde-functionalized glass surface followed by photopolymerization by using a 254 nm light source. To measure the biotin-streptavidin binding, we conjugated photoactivatable biotin to immobilized PCDA-NH(2) liposomes by UV irradiation (365 nm) and subsequently allowed them to interact with streptavidin. We analyzed the fluorescence using a fluorescence scanner and observed single liposomes using NSOM. The average height and NSOM signal observed in a single liposome after binding were similar to 31.3 to 8.5 +/- 0.5 nm and 0.37 to 0.16 +/- 0.6 kHz, respectively. This approach, which has the advantage of not requiring a fluorescent label, could prove highly beneficial for single molecule detection technology.