The aim of our study was to investigate the use of targeted ultrasound microbubbles (MBs) for molecular imaging of murine endothelial CD81 expression. In the study, the anti-CD81-coated MBs was successfully prepared and characterizated. Murine bEnd.3 cells were stimulated with phenazine methosulfate (PMS) to induce the up-regulation of CD81 expression. Changes in CD81 expression after stimulation were tracked with anti-CD81-coated MBs and imaged by using SONIX RP ultrasound imaging system. Our results showed that endothelial CD81 expression was gradually up-regulated with the increase of PMS concentration. Correspondingly, the accumulation of targeted MBs was also gradually improved and could be inhibited competitively. The mean video intensity of stimulated cells from backscatter of the CD81-targeted MBs was significantly higher than that of the non-stimulated control (mean ± SD: 17.5 ± 3.6 versus 12.1 ± 2.9 pixel intensity; P < 0.01). In conclusion, CD81-targeted MBs allows non-invasive assessment of the expression levels of CD81 on the bEnd.3 cells and may provide potential insights into early atherosclerotic plaque detection and treatment monitoring using molecular ultrasound imaging.