We introduced a promising patterned substrate by using a microcontact printing method that can be used for SERS immunoassays based on antigen-antibody binding. SERS spectrum of the Raman reporter with antibody, which is rhodamine 6G (R6G) adsorbed on colloidal gold nanoparticles, was observed only for the surfaces in which prostate-specific antigen (PSA) is present on the substrate that is attached to an immobilized layer of antibody on the gold nanoparticles layer of the patterned substrate. Raman mapping images clearly showed that the antibodies on the Raman reporter were successfully and selectively conjugated with the antigen on the patterned substrate. This method could be potentially extended to multi-protein detections and ultrasensitive biosensors.