Objective To investigate the therapeutic effects of low-frequency ultrasound combined with 5-fluorouracil(5-FU) nanobubbles on post radiofrequency ablation (RFA) hepatocellular carcinoma (HCC) nude mice. Methods A xenograft HCC mouse model was established in 60 BALB/c nude mice (6-8 weeks) using human hepatocellular carcinoma cell lines (HepG2).The mice were randomized into four groups after RFA ablation of 80%: saline group, 5-fluorouracil loaded nanobubbles (5-FU)group, non-low frequency ultrasound irradiated 5-FU-loaded nanobubbles (non-low frequency ultrasound + 5-FU) group, and low-frequency ultrasound irradiated 5-FU-loaded nanobubbles (low-frequency ultrasound + 5-FU) group. The nude mice of the last three groups received a tail vein injection of 5-FU loaded nanobubbles (0.1 μg/μl), 200 μl each time, once every three days, and three consecutive injections. The low-frequency ultrasound + 5-FU group received additional irradiation of low-frequency ultrasound (1 MHz, 2 W/cm²) for 5 min after being injected into the nanobubble. The growing status, tumor size and survival time of nude mice bearing tumor in each group were observed and compared. At the end of the treatment, the tumor tissue was taken, apoptosis index was detected through the TUNEL method, and the tumor neovascular density (MVD) was detected by the CD34-MVD method. The apoptosis index and MVD values were compared in each group. Results After treatment, the nude mice in the different groups exhibited weight loss, a reduction in activity, appetite loss, and other symptoms to varying degrees. However, the growing status of tumor-bearing nude mice in low-frequency ultrasound + 5-FU group was significantly better than the other three groups. The tumor volume of nude mice in each group gradually increased. The tumor growth rate of low-frequency ultrasound+ 5-FU group was significantly lower than that of the other three groups, and the difference was statistically significant (P<0.05).The survival time of tumor-bearing nude mice in low-frequency ultrasound+5-FU group was significantly longer than that of the other three groups, and the difference was statistically significant (P<0.05). The apoptotic index of tumor cells in each group: low frequency ultrasound + 5-FU group (43.2%±4.4%) > non-low frequency ultrasound + 5-FU group (31.3%±4.3%) > 5-FU group(20.7%±2.9%) > saline group (10.8%±2.4%), and there were statistically significant in each group (P<0.05). Meanwhile, except for the low-frequency ultrasound + 5-FU group, multiple positive staining areas were seen in the tumors of the other three groups, and there were more positively stained tumor tissues. The MVD values (piece/HP) of each group: low frequency ultrasound + 5-FU group (8.9±1.3) < non-low frequency ultrasound + 5-FU group (20.1±3.2) <5-FU group (25.0±4.2) <saline group (29.9±2.0), and there were statistically significant in each group (P<0.05). Conclusions 5-fluorouracil loaded nanobubbles combined with low-frequency ultrasound could further improve drug targeting, effectively inhibit the growth of HCC, which can significantly inhibit residual cancer cells after RFA.