We report the self-spreading behavior of single lipid bilayers containing 7-nitrobenz-2-oxa-1, 3-diazol-4-yl (NBD) fluorophores on a patterned surface equipped with a single nanogap. We found that the behavior of the fluorophores around the nanogap depended on the position of the dyes in the lipid molecules. For acyl chain-labeled lipids, the fluorescence intensities decreased discontinuously before and after they passed through the nanogap, whereas little decrease was observed for a headgroup-labeled lipid. A possible mechanism can be explained in terms of the reorientation of dyes in a lipid bilayer. Acyl chain-labeled NBD fluorophores are known to loop back to a lipid-water interface in a lipid bilayer. Since this effect makes an effective size of the lipid molecule larger, dye molecules easily suffer from steric hindrance when they pass through a nanogap leading to the fluorescence intensity reduction.