This paper proposes a method for detecting contrast agents in ultrasound image sequences to develop diagnostic ultrasound imaging systems for tumor diagnosis. The conventional methods are based on simple subtraction of ultrasound images to detect ultrasound contrast agents, where the conventional methods need ultrasound image sequences with and without contrast agents. Even if the subject slightly moves, the detection result of the conventional methods includes significant errors. On the other hand, the proposed method employs the spatio-temporal analysis of the pixel intensity variation over several frames. The proposed method also employs motion estimation to select optimal image frames for detecting contrast agents. Through a set of experiments using mice, we demonstrate that the proposed method exhibits efficient performance compared with the conventional methods.