Objective To investigate the effect of low-frequency low-intensity ultrasound irradiation microbubbles on drug resistance in triple-negative breast cancer MDA-MB-231/DOX cells. Methods The optimal irradiation time of LILFU-MB was optimized. The MDA-MB-231/DOX cells were exposed to various concentrations of Doxorubicin (DOX) for 24h (ranging from 10-2μM to 103μM) to assess the half-inhibitory concentration (IC50) of DOX. Four groups were formed: control group, DOX group, LILFU-MB group and DOX+LILFU-MB group. Cell proliferation, migration, apoptosis, and protein expression levels of P-gp, Bcl-2, Cyt-c and Caspase-3 were evaluated. Results After a non-toxic irradiation period of 30s with LILFU-MB treatment reduced the IC50 value of DOX in drug-resistant cells. Compared to individual treatments with either DOX or LILFU-MB alone, the combination treatment in the DOX+LILFU-MB group exhibited significantly lower proliferation rate and migration rate as well as more pronounced apoptotic karyotype changes. Furthermore, this combined treatment resulted in increased apoptosis rate , elevated protein expression levels of Cyt-c and Caspase-3 (P<0.05) and decreased protein expression levels of Bcl-2 (P<0.05). Importantly, the expression level of P-gp protein was significantly down-regulated by the DOX+LILFU-MB group (P<0.05).