In this paper, the design procedure of an auto-focusing device for a microscope is presented. To obtain a long focusing distance, a new flexure positioning mechanism is devised based on multi-stage leaf springs. The proposed device allows a precise focusing without friction and backlash effects. A voice coil motor is employed to drive the positioning mechanism. The stiffness model and resonant-frequency model are developed analytically. These models are employed for an architectural optimization of the mechanism parameters to maximize the resonant frequency under the driving stroke and force constraints. The performance of the designed flexure mechanism is validated through finite-element analysis (FEA) simulation investigations. Results show that the positioning mechanism enables a long focusing range over 10 mm with a high resonant frequency over 450 Hz, which allows an auto-focusing operation with rapid response. Moreover, the performance of the auto-focusing device is validated through experimental studies on a fabricated prototype.