We propose a prototype of silicon-on-glass microchip for protein detection by bead-based affinity chromatography. The microchip has five channels integrated by composing one beads reactor per one channel. Especially, an effective protein analysis mechanism is presented where the three protein-pretreatment processes are simultaneously performed on a single beads reactor: selective detection (purification / sensing), pre-concentration and protein digestion. Since the five channels are closely spaced in parallel on the microchip, it is possible to inspect the five different detection results on real-time in a single microscope image. The microchip is fabricated on silicon-on-glass (SiOG) to make a mechanically strong and vertically transparent structure for efficient fluid interconnection and fluorescence detection, respectively. Within the microchip, the grid-type filter is formed on channel output to physically trap 38 ~ 50 μm diameter microbeads. The dimension of one grid is 30 × 30 μm2. The volume flow rate was investigated experimentally on the case of bead-packed chamber, and the resulted value was compared to that of the case of hollow chamber. In this research, we used self-cleavage free aptazymes as detection ligands immobilized on polystyrene microbeads. The target proteins are firstly on-chip concentrated and fluorescence-detected (confocal microscopy), and secondly checked off-chip by using MALDI-TOF. If the two analyses are used cooperatively, it is expected that the accuracy in diagnostic analysis will be enhanced in biosensing system. Especially by using this free aptazymes system, we don't need to consider the requirement of fluorescence tagging and the difficulty of eluting antibody-bound proteins from microbeads without bad effects of harsh elution conditions in protease treatment. We analyzed the on-bead detection of HCV replicase and HCV helicase respectively by measuring fluorescence intensities at different concentrations, and also performed a selectively detection of HCV helicase from protein mixtures.