Objective: To study on the preparation process of a new surfactant-based microbubble ultrasound contrast agent and to evaluate its contrast effects in vivo. Methods: Microbubble ultrasound contrast agent with three ester surfactants and other additives as its shell materials was prepared by sonication. Sulfur hexafluoride was adopted as the inner gas of the microbubbles. New methods through the combination of optical microscope and some softwares were used to measure the size distribution and the concentration of the microbubbles. Some parameters such as the pH value of the phosphate buffer, quantity of the carboxylic methyl cellulose in the shell materials, selection of the ultrasound power and process time, were studied. Six hybirded dogs were used to verify the in vivo contrast imaging of the contrast agent using second harmonic power Doppler modality. Safety and persistent time of the agent inner animal body were also investigated. Results: Ultrasound contrast agent prepared in the experiment had an average microbubble diameter of 3.95 microns with concentration of 3.6×109 microbubbles per millilitre. Carboxylic methyl cellulose was found as an important shell material which had obviously effect on the microbubble stability and production even with a little quantity. The buffer pH value also had a key role on the microbubble formation and the final production. When the buffer pH value reached 7.4, there was no microbubble produced. Under the approximate microbubble production, process time could be shorten with the increasing ultrasound power. The obvious ultrasound contrast imaging effects were detected in the dogs heart chamber and liver as well as kidney using only one millilitre agent when diluted. The agent was found safe to the dogs. At the same time, persistent time of the agent was found over 20 min in the dogs body. Conclusion: The new ultrasound contrast agent prepared in the experiment has high microbubble production and concentration, narrow microbubble size distribution ranging in several microns, well stability, little dosage needed in the contrast, well safety to the dogs and long persistent time, obvious contrast imaging effect in the dogs heart chamber, kidney and liver. These experiment data indicate that the new ultrasound contrast agent with three ester surfactants and carboxylic methyl cellulose as its main shell materials can be further developed for clinical purposes.