Three wheat varieties were selected as experimental materials to measure the activity of polyphenol oxidase According to the method from Measurement of Polyohenol Oxidase Activity in Wheat Flour in national standard draft. And found it has three deficiencies: first, because of the lack of crude extcation of polyphenol oxidase, it led to the less quantity of polyohenol oxidase, and couldn’t reflect the real state of the activity of polyphenol oxidase; second, because of the unsuitable reaction temperature, it couldn’t help the enzyme reaction completely; third, adding catechol before centrifuge made a part of product intercepted by precipitation, thus it has poor stability and exhibited small activity of polyphenol oxidase. Based on this method, explored the influences of different factors on the determination of activity of the polyphenol oxidase to optimize the method, including the extraction time, the heat temperature, and the time for adding substrate. The results revealed of the optimum method seems to be better than before, the optimum process were: extraction 4 h, add catechol after centrifuge, heat at 70 ℃ in 15 min, and could get higher activity of polyphenol oxidase and had better repeatability, it was more reliable and stable. Also detected 50 wheat varieties by these two methods, and divided special noodles flour into different level by the activity of polyphenol oxidase.