Objective: To investigate the anti-tumor effect of the transfection of plasmid containing CD/TK double suicide genes mediated by microbubble-enhanced ultrasound exposure on breast cancer in vivo. Methods: After the xenograft tumor model was created in nude mice using human breast cancer MCF-7 cells, the tumor-bearing mice were randomly divided into control group, plasmid group, ultrasonic irradiation group and ultrasonic irradiation plus microbubble agent group, with 5 mice in each group. Mice in control group were only given the corresponding CD and TK prodrugs (5-fluorocytosine and ganciclovir), mice in plasmid group were injected with the plasmids (containing CD/TK gene) and prodrugs, mice in ultrasonic irradiation group were injected with plasmids and prodrugs combined with ultrasonic irradiation, and those in ultrasonic irradiation plus microbubble agent group were injected with targeted ultrasonic contrast agent (mixture of plasmids and microbubble agent) combined with ultrasonic irradiation. The growth of the tumors was recorded during the experiment. After the tumors were enucleated at post-treatment day 5, the tumor inhibition rate in each group was calculated, the transfection of the target genes in the tumors was observed by an inverted fluorescence microscope, and the transfection efficiency was also analyzed; the expression of the target genes was determined by RT-PCR method; the microvessel density (MVD) of the tumors was detected by immunohistochemical staining. Results: Compared with control group, the tumor growth in plasmid group showed no obvious difference (P>0.05), while in both ultrasonic irradiation group and ultrasonic irradiation with microbubble agent group it was significantly inhibited (both P<0.05), and tumor inhibition rate in plasmid group, ultrasonic irradiation group and ultrasonic irradiation plus microbubble agent group was 3.72%, 21.40% and 47.13%, respectively. The gene transfection efficiency in plasmid group, ultrasonic irradiation group and ultrasonic irradiation plus microbubble agent group was 0.78%, 2.81% and 23.87%,respectively, which in the latter group was significantly higher than that in the former two groups (both P<0.05). The positive DNA fragment of CD/TK gene was present in the tumors from the ultrasonic irradiation group or ultrasonic irradiation plus microbubble agent group, but was absent in those from control group and plasmid group. The MVD count in tumors from both ultrasonic irradiation group and ultrasonic irradiation plus microbubble agent group was significantly lower than that in control group (both P<0.05), which in ultrasonic irradiation with microbubble agent group was even lower than that in ultrasonic irradiation group (both P<0.05), but showed no statistical difference between plasmid group and control group (P>0.05). Conclusion: The double suicide gene system mediated by microbubble-enhanced ultrasonic irradiation can effectively increase the transfection efficiency and expression of the target gene, and thereby exert an enhanced inhibitory effect on tumor growth and angiogenesis.