A binary optical encoding strategy is proposed to meet the increasing requirements of multiplex bioassays. As illustrated in fluorescence immunodetection of multiplex antigen molecules, photonic crystal beads (PCBs) and quantum dots (QDs) can be used as biomolecular microcarriers and fluorescence labels, respectively. The categories of antigens were deciphered by the binary combination of optical spectra of PCBs and QDs as independent encoding elements. The number of categories that could be detected was theoretically m x n, where m and n represent the number of encoding PCBs and QDs, respectively. In addition, the concentrations of the antigens were determined by the fluorescence signals of the QDs. Results of sensitivity analysis indicate that a low-level detection of 58 pg/mL was achieved. Because of the special nanostructures of these two encoding elements, the binary encoding strategy demonstrated its superiority and practicability when compared with single PCB or QD encoding. This supports potential application in multiplex bioassays.