Abstract: According to the problems of low degree of automation, slow process of focusing and high requirement of operator experience caused by traditional manual focusing microscope, a new optical microscope system based on depth from focus was proposed. Firstly, according to the general microscope structure with auto-focusing based on image processing, a set of microscope system was designed, constituted of ARM development board, touch-sensitive LCD, Arduino UNO development board, infrared tube, industrial digital microscope and stepping motor, etc. Then, a focusing window selection method combining regions of interest with different sizes was proposed based on the characteristics of touch-sensitive LCD screen.And an improved method with fault tolerant was put forward based on the mountain climb searching algorithm and the common gradient function as the image definition evaluation function. Furthermore, the real-time, sensitivity and fault-tolerant performance for different image definition evaluation functions and sizes for selection window were tested in the experiment.The results show that the system has high reliability.