The phase-separated structure of poly(isobutyl methacrylate)-block- poly(octadecyl metha-crylate) (PiBMA-b-PODMA) monolayer and single poly(isobutyl methacrylate) (PiBMA) chain dispersed therein were investigated by scanning near-field optical microscopy (SNOM), which enabled us to observe with high spatial resolution beyond the diffraction limit of light. The conformation of the individual homo-PiBMA chains was quantitatively evaluated from the fluorescence intensity distribution. We found that the homo-PiBMA chains were almost located at the center of the PiBMA domain. The homo-PiBMA chain was oriented itself depending on its location in the PiBMA domain. When the homo-PiBMA chain locates at the center of the PiBMA domain, it tends to orient itself in the direction parallel to the interface between the two microdomains. When the homo-PiBMA chain locates near the interface, it orients itself in the direction perpendicular to the interface. © 2009 American Chemical Society.