Objective To investigate the effect of diagnostic ultrasound with different duty cycle combined with microbubbles on blood perfusion of tumor with lack of blood supply in rats. Methods Forty five wistar rats were selected to establish a rat C6 glioma model subcutaneously on the right back. According to different pulse time and interval time, they were randomly divided into 5 groups with 9 rats in each group: group A, pulse time 5s, interval time 1s; group B, pulse time 1s, interval time 1s; group C, pulse time 1s, interval time 5s; group D, pulse time 5s, interval time 5s; group E, sham explore with ultrasound. The corresponding treatment pulse duty cycles of each group were 0.167%, 0.1%, 0.033%, 0.1% and 0, respectively. Each group of tumor bearing rats were treated with ultrasound combined with microbubbles/sham explore with ultrasound for 10 minutes. Contrast-enhanced ultrasound was performed before and immediately after treatment to obtain the time intensity curve (TIC), the peak intensity (PI) and area under the curve (AUC) of each group were compared; The contrast-enhanced ultrasound images of tumors in each group were compared and analyzed, and the contrast-enhanced ultrasound visual score was performed; Three rats in each group were randomly selected, and the tumor tissues were stained with HE to observe the pathological changes after ultrasonic treatment/sham ultrasonic explore. Results Compared with before treatment, the AUC value of group A and group B increased after treatment, and the difference was statistically significant (t=-2.726, -3.922; P= 0.026, 0.004). There were no significant differences in PI value and AUC value of group C, group D and group E after treatment (all P > 0.05). Compared with group C, group D and group E, the visual score of contrast-enhanced ultrasound in group A and group B was higher, the difference was statistically significant (all P<0.05), and there were no significant differences among the other groups (all P>0.05). Obvious vasodilation was observed in group A and group B, a small amount of red blood cell exudation was observed in some perivascular tissues in group A, and there was no obvious vasodilation in group C, group D and group E. Conclusion Low intensity diagnostic ultrasound combined with microbubbles with pulse time of 5s, interval time of 1s, duty cycle of 0.167% and pulse time of 1s, interval time of 1s and duty cycle of 0.1% can enhance the blood perfusion of tumor with lack of blood supply. The latter is a relatively safe acoustic parameter for ultrasonic treatment.