We have developed a novel multiplexed bead-based mesofluidic system (MBMS) based on the specific recognition events on the surface of a series of microbeads (diameter 250 μm) arranged in polydimethylsiloxane (PDMS) microchannels (diameter 300 μm) with the predetermined order and assembled an apparatus implementing automatically the high-throughput bead-based assay and further demonstrated its feasibility and flexibility of gene diagnosis and genotyping, such as β-thalassemia mutation detection and HLA-DQA genotyping. The apparatus, consisting of bead-based mesofluidic PDMS chip, liquid-processing module, and fluorescence detection module, can integrate the procedure of automated-sampling, hybridization reactions, washing, and in situ fluorescence detection. The results revealed that MBMS is fast, has high sensitivity, and can be automated to carry out parallel and multiplexed genotyping and has the potential to open up new routes to flexible, high-throughput approaches for bioanalysis.