In this study, labeled purple membrane (PM, Halobacterium salinarium) patches with maintained bioactivity were prepared by biotinylation of the glycolipids on the extracellular (EC) surface of the PM. After in situ streptavidin incubation, the extracellular surface could be directly visualized because it was completely covered with bright dots. The height of a single biotin/streptavidin interaction layer was approximately 2.5 nm. By using calcium thioglycolate-modified tips and an atomic-force microscope (AFM), two distinct topographical features of PMs placed in a buffer with low salt concentration were recognized: the flat EC surface and the domelike cytoplasmic (CP) surface. Biotinylation and AFM with modified tips were simultaneously used with consistent results. Those observations are useful for future studies on PM bioconjugation and oriented assembly as well as the design of bacteriorhodopsin-based photoelectric devices.