The purpose of this study was using a developed microfluidic chip to prepare size-controlled monodisperse chitosan microparticles encapsulating ampicillin. Our strategy is that a chitosan aqueous solution (the disperse phase) is fed into the microfluidic chip equipped with a cross-junction microchannel, and is sheared by the viscous oil flows (the continuous phase) to form monodisperse semi-product, chitosan emulsions. These fine emulsions are then gelled into stability upon gelation by injection of copper sulfate solution at the terminal microchannel of the microfluidic chip, and finally the uniform chitosan microparticles are formed in an efficient manner. The proposed chip is fabricated by a CO(2) laser machine on a conventional poly methyl methacrylate (PMMA) substrate. This microfluidic chip has four inlet ports, one cross-channel and one outlet port. We have demonstrated that one can control the size of chitosan microparticles from 100 to 800 microm in diameter (with a variation less than 5%) by altering the relative sheath/sample flow rate ratio. Experimental data showed that when given a steady continuous phase (oil flow), the emulsion size increases with the increase in average velocity of the dispersed phase flow (sample flow). In addition, the release of the model drug (ampicillin) from these microspheres is proved to be once-daily for clinical application. We also revealed that appropriate particle sizes for different release patterns are predictable, enabling better applications of chitosan as a drug carrier.