We present a large-area hemispherical perforated microwell structure for a bead based bioassay. The fabrication process for the hemispherical perforated microwell consisted of three steps: the convex micropatterned mold production from the concave micropatterned silicon wafer, hot embossing to generate the concave micropattened acrylate sheet, and reactive ion etching to make the bottom holes. We applied the hemispherical perforated microarray for the bead based aptamer screening, in which the bead trap, the in-situ fluorescence monitoring of aptamer-target protein interaction on the beads, the retrieval of the bioassay beads, and characterization of the aptamer by MALDI-TOF mass spectrometry could be sequentially executed.