A safe, effective, and inclusive gene therapy will significantly benefit a large population of hemophilia patients. We employed a minimally invasive transcutaneous ultrasound mediated gene delivery (UMGD) strategy combined with microbubbles (MBs) to enhance gene transfer into four canine livers. A high-expressing, liver-specific human factor VIII (hFVIII) plasmid/MBs mixture was injected into the hepatic vein via balloon catheter under fluoroscopy guidance with simultaneous transcutaneous UMGD treatment targeting a specific liver lobe. Therapeutic levels of hFVIII expression were achieved in all four dogs and hFVIII levels were maintained at a detectable level in three dogs throughout the 60-day experimental period. Plasmid copy numbers correlated with hFVIII antigen levels and plasmid-derived mRNA was detected in treated livers. Liver transaminase levels and histology analysis indicated minimal liver damage and a rapid recovery post-treatment. These results indicate that liver-targeted transcutaneous UMGD is promising as a clinically feasible therapy for hemophilia A and other diseases.