Objective：To prepare gene loaded nanaobubbles and explore their effects of ultrasonography and targeted killing of hepatocellular carcinoma cells in vivo and in vitro. Methods：Positively charged nanobubbles with the peripheml phospholipid shells filled by suphurhexafluoride（SF6）gas were fabricated through the method of lipid thin-film，hydration and mechanical agitations. Then simplex herpes virus thymidine kinase（HSV-TK）plasmid DNA coupled with alpha fetoprotein（AFP）promoter was conjugated to create gene loaded nanobubbles. The characteristics such as morphology，average particle size，zeta potential，capacity of DNA binding and gene transfection were detected. Then the effect of ultrasound contrast enhancement imaging was investigated in vivo and in vitro. Finally，the killing efficiency of gene-loaded nanobubbles on BEL-7402 and SMCC-7721 cells when combined with ultrasound-targeted microbubble destruction（UTMD）was detected using CCK-8 and flow cytometry（FCM）assay. Results：The nanobubbles appeared as a spherical morphology with a core-shell structure under TEM，and they were about 667 nm in mean hydrodynamic diameter with positive charge. Nanobubbles can effectively bind with plasmid DNA when particle/DNA weight ratio was set at 5∶1. The gene-loaded nanobubbles had good ultrasonic contrast enhancement characteristics in vitro and in vivo ultrasonography. RT-PCR assays indicated that nanobubbles combined with UTMD enabled HSV-TK to be expressed in AFP positive hepatocellular carcinoma cells rather than AFP negative ones. The analysis of CCK-8 and FCM showed that gene-loaded nanobubbles combined with UTMD could significantly inhibit the proliferation of BEL-7402 cells and induce apoptosis，which was significantly different from other groups（P < 0.05）. Conclusion：Gene-loaded nanobubbles with good ultrasonic imaging function can effectively induce targeted killing of AFP positive hepatocellular carcinoma cells.