Automatic optical inspection plays a key role in inspection of automations and equipments. It can replace manual inspect, reduce the production costs and achieve high speed and high precision with the inspection. Increasing the quality and efficiency of production materially. Mura defect automatic optical inspection system combines with electro-mechanical Systems, image acquisition systems and inspection software. In inspection software, it is to implement polynomial background surface estimation, use the standard which was defined by SEMI and apply auto-focus method to ensure the quality of image. This system has 100% accuracy and spent 45∼55 seconds in the experimental stage. There are two options can be selected, inline or off-line inspection.