Polyclonal antibodies are found to preferentially bind to the protein molecules on the edge of a protein pattern before their binding to the interior protein molecules. Highly ordered lysozyme patterns were fabricated on carboxylic acid-terminated chemical templates on the octadecyltrichlorosilane (OTS) self-assembled monolayer surface. Polyclonal anti-lysozyme antibodies reacted with the immobilized lysozyme pattern at different exposure levels. The binding of antibodies towards lysozyme patterns was directly investigated by the Atomic Force Microscope (AFM). Protein molecules on the pattern edge always bind to the antibodies before the protein molecules in the center of the pattern. In addition, the topography of the immobilized protein pattern affects the antibody binding direction. We found that the antibodies bound to the edge lysozyme molecules of a half-buried pattern from the top whereas they bound to the edge lysozyme molecules of an extruding protein pattern from the side. We explain the observed differences in antibody binding by the different spatial accessibilities of the edge protein and the interior protein in a protein pattern. The “edge effect” potentially can lead to an effective means to improve the activity of the immobilized enzymes.