Abstract: Based on the theoretical motion equation of encapsulated microbubbles in the ultrasound field, the subharmonic characterizations of microbubbles were optimally designed and analyzed by a computer aided design system. The effects of size, shell elasticity and acoustic pressure on the subharmonic response of microbubbles were calculated theoretically to obtain the better parameters for nondestructive subharmonic imaging. In addition, the microbubbles with different shell elasticity were prepared, and their subharmonic responses were measured in vitro. The results of theoretical calculation and acoustic measurement showed that good subharmonic enhancement can be obtained by using the encapsulated microbubbles with the mean size of 3μm, which were prepared from the surfactant solution with the proper ratio of shell material. It is also shown that the best operating acoustic pressure is 200~400 kPa for nondestructive subharmonic imaging based on such kind of microbubbles.