Usability testing is a very important step in improving App design and development. The traditional usability testing methods are based on users'expressions and behaviors, which hardly show users' emotional experience and cognitive load in real time. The introduction of an electrophysiological technique can make up for the deficiency of the traditional usability testing methods. In this study, a usability testing was carried out with the old and the new version of an App software. The behavior and the subjective evaluation of the participants were recorded, and their GSR and ECG signals were collected. Then, 14 physiological characteristics, such as GSR-Mean, LF, HF, LF/HF, etc., were extracted from the GSR and ECG signals. These characteristics were analyzed, and a significance test of difference of the two versions was made. This research indicated that there is a certain application value of GSR and HRV in usability testing and evaluation of an App product. But the meanings of the physiological characteristics must be explained in combination with the behavior and subjective evaluation of users. The result can prove that physiological characteristics have obvious advantages in real-time monitoring users' emotional changes, which can be helpful to find the usability problems of the product.