Janus particles with anisotropic biofunctionalities are perfect models to mimic anisotropic architectures and directional interactions that occur in nature. It is therefore highly desirable to develop reliable and efficient methods to synthesize biofunctional Janus particles. Herein, a facile method combining seeded-emulsion polymerization and thiol-click chemistry has been developed to synthesize Janus particles with glucose moieties on one side. These biofunctional Janus particles show region-selective binding of protein, which represents a big step toward biomimicry, and demonstrates the potential of the bioJanus particles for targeted drug delivery and binding.