An easy preparation method of multilayer fluorescence optically encoded beads for protein detection is presented. The beads, which consist of multicolored layers, are made from amino polyethylene glycol grafted polystyrene (PS-g-PEG) beads by using several fluorescent dyes such as fluorescein isothiocyanate (FITC) and rhodamine via controlling diffusion of an Fmoc-protecting group after HCl solution swelling. A biotin, glutathione S-transferase (GST) antibody, and an RNA aptamer that specifically recognize streptavidin, GST antigen, and hepatitis C virus (HCV) helicase are introduced to the optically encoded beads and monitored for their binding activity to the target molecules. After binding, the ligands are identified easily by their color codes.