We report a novel optoelectrofluidic immunoreaction system based on electroosmotic flow for enhancing antibody–analyte binding efficiency on a surface-based sensing system. Two conventional indium tin oxide glass slides are assembled to provide a reaction chamber for a tiny volume of sample droplet (∼5 μL), in which the top layer is employed as an antibody-immobilized substrate and the bottom layer acts as a photoconductive layer of an optoelectrofluidic device. Under the application of an AC voltage, an illuminated light pattern on the photoconductive layer causes strong counter-rotating vortices to transport analytes from the bulk solution to the vicinity of the assay spot on the glass substrate. This configuration overcomes the slow immunoreaction problem of a diffusion-based sensing system, resulting in the enhancement of binding efficiency via an optoelectrofluidic method. Furthermore, we investigate the effect of optically-induced dynamic AC electroosmotic flow on optoelectrofluidic enhancement for surface-based immunoreaction with a mathematical simulation study and real experiments using immunoglobulin G (IgG) and anti-IgG. As a result, dynamic light patterns provided better immunoreaction efficiency than static light patterns due to effective mass transport of the target analyte, resulting in an achievement of 2.18-fold enhancement under a growing circular light pattern compared to the passive mode.