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This study presents a new method for generating tunable and uniform magnetic alginate microspheres, which can be widely employed in the field of the bead-based bio sample pretreatment and enrichment process during the clinical diagnoses. A microfluidic device comprising microchannels and controllable moving-wall structures was fabricated by using MEMS processes. The key feature of the controllable moving-wall structure is located nearby the flow-focusing microchannels and can be fine-tuned by the pneumatic pressure such that the sheath flows can be locally accelerated. With this approach, the size of the alginate microspheres can be fine-tuned without adjusting the syringe pumps. Therefore, the uniform magnetic alginate microspheres can be successfully generated.