We performed a direct immunoassay inside a microfluidic channel on patterned streptavidin-coated beads, which captured fluorescently-labeled biotin target molecules from a continuous flow. We arranged the beads in a dot array at the bottom of the channel and demonstrated their position- and flow rate-dependent fluorescence. As the target analyte gets gradually depleted from the flow when passing downstream the channel, the highest fluorescence intensity was observed on the most upstream positioned dot patterns. We propose a simple analytical convection model to explain this spatio-temporal fluorescence.