This work was to report preparation and quality control of targeted microbubbles (MB(t)) via biotin-avidin bridge, specifically verification of the "biotin-avidin-biotin" sandwich structure. (1) Lipid microbubbles filled with sulphur hexafluoride were produced by sonication and compared with commercially available microbubbles. (2) MB(t) were produced via biotin-avidin bridge. Specifically, the "biotin-avidin-biotin" sandwich structure on the surface of MB(t) was verified in vitro using fluorescence. (3) Adhesion of alpha(v)beta(3)-integrin targeted MB(t) (MB(αvβ3)) to human umbilical vein endothelial cells (HUVECs) was tested using the parallel plate flow chamber (PPFC). "Biotin-avidin" is a reliable method to attach molecular probe onto the surface of microbubbles.