Anisotropic microarchitectures with different physicochemical properties have been developed as advanced materials for challenging industrial and biomedical applications including switchable displays, multiplexed biosensors and bioassays, spatially-controlled drug delivery systems, and tissue engineering scaffolds. In this study, anisotropic biohybrid microparticles (MPs) spatio-selectively conjugated with two different antibodies (Abs) are first developed for fluorescence-based, multiplexed sensing of biological molecules. Poly(acrylamide-co-acrylic acid) is chemically modified with maleimide- or acetylene groups to introduce different targeting biological moieties into each compartment of anisotropic MPs. Modified polymer solutions containing two different fluorescent dyes are separately used for electrohydrodynamic co-jetting with side-by-side needle geometry. The anisotropic MPs are chemically stabilized by thermal imidization, followed by bioconjugation of two different sets of polyclonal Abs with two individual compartments via maleimide-thiol coupling reaction and Huisgen 1,3-dipolar cycloaddition. Finally, two compartments of the anisotropic biohybrid MPs are spatio-selectively associated with the respective monoclonal Ab-immobilized substrate in the presence of the antigen by sandwich-type immunocomplex formation, resulting in their ordered orientation due to the spatio-specific molecular interaction, as confirmed by confocal laser scanning microscopy. In conclusion, anisotropic biohybrid MPs capable of directional binding have great potential as a new fluorescence-based multiplexing biosensing system.