Objective: To study on the preparation process of a new surfactant-based microbubble ultrasound contrastagent and to evaluate its contrast effects in vivo. Methods: Microbubble ultrasound contrast agent with three ester sur-factants and other additives as its shell materials was prepared by sonication. Sulfur hexafluoride was adopted as the in-ner gas of the microbubbles. New methods through the combination of optical microscope and some softwares were usedto measure the size distribution and the concentration of the microbubbles. Some parameters such as the pH value of thephosphate buffer, quantity of the carboxylic methyl cellulose in the shell materials, selection of the ultrasound powerand process time, were studied. Six hybirded dogs were used to verify the in vivo contrast imaging of the contrast agentusing second harmonic power Doppler modality. Safety and persistent time of the agent inner animal body were also in-