In this study, an ultrasound microbubble contrast agent with integrin αvβ3 as the target was prepared. It was used for the target-finding diagnosis of breast cancer cells. The sulfhydryl group of iRGD peptide and the maleimide group of DSPE-PEG2000 Maleimides were connected by a thin-film hydration method to obtain a new type of ultrasound microbubble contrast agent (iRGD-DSM). First, a particle size analyzer and an optical microscope were used to characterize the iRGD-DSM. Then, the mouse breast cancer cell 4T1 was used for breast cancer ultrasound diagnosis by in vitro culture, and the binding of the contrast agent to the cells was analyzed. Also, quantitative analysis was performed by flow cytometry. The test results showed that an iRGD peptide, iRGD/DSPE-P2000, was obtained based on the chemical coupling of sulfhydryl-maleimide groups. In terms of average particle size, microbubble distribution, and concentration, the iRGD-DSM contrast agent prepared in this study was similar to conventional microbubble contrast agents. Also, when the DSPC, DSPEPEG, iRGD/DSPE-P2000 molar ratio is 36:3:1, higher peptide content can be obtained. Dynamic/static specific targeting of vascular endothelial cells (bEND.3) adhesion test confirmed that iRGD-DSM contrast agent can be firmly combined with bEND.3. In the target-finding test of breast cancer cells 4T1, the ultrasound microbubble contrast agent proposed based on this study had obvious binding imaging with breast cancer cells 4T1. In numerical analysis, the binding rate of the contrast agent to 4T1 cells reached (95.75 ± 1.43)%. Therefore, it was confirmed that the ultrasound microbubble contrast agent iRGD-DSM with integrin αvβ3 as a target has certain in vitro targeting ability for breast cancer diagnosis.