We present a simple synthetic approach for the preparation of monodisperse thermosensitive gelatin microspheres in a microfluidic system. Based on the mechanism of shear force-driven break-off, aqueous droplets of a gelatin solution were continuously produced in an immiscible continuous fluid. Under cooling conditions, the gelatin droplets solidified into hydrogel microspheres, which resulted from the aggregation or crystallization of collagen folds. The produced gelatin microspheres possess a high monodispersity and fast response to environmental temperature. In addition, the size of the prepared microspheres can be manipulated by altering the flow rate of the continuous phase or aqueous phase, and the physical strength of the gelatin microspheres can be controlled by simply changing the gelatin concentration. Furthermore, this approach enables the preparation of monodisperse microspheres with the ability to exhibit different thermosensitivities and encapsulate colloidal particles under mild conditions, which demonstrate sequential release of the desired encapsulants according to the responsive temperature.