Abstract: Objective To identify best local ultrasound transfection conditions in rats with injured achilles tendons and granulation tissues. Methods Rat models with bilateral injured achilles tendons were established. A mixture of microbubbles and enhanced green fluorescent protein (EGFP) plasmids were injected into the achilles tendons. The injured tendons were subject to ultrasound irradiation under different output intensity, duty cycle and irradiation time. The rats were divided into four groups: ①plasmid+microbubbles+ultrasound (PL+MB+US); ②plasmid + microbubble (PL+MB); ③plasmid + ultrasound (PL+US); ④ plasmid only (PL). We identified the best ultrasound conditions based on the highest transfection efficiency in the achilles tendons and granulation tissues and the least normal tissue damage. Results Under the condition of 2 W/cm2 power output and 20% duty cycle of ultrasound irradiation for 10 min, EGFP expression was demonstrated in the achilles tendons and granulation tissues without showing significant damage to normal tissues. Higher levels of EGFP expression were found in the group of PL+MB+US than the other groups (no significant differences in EGFP expression were found among the other three groups). Conclusion Under proper conditions, ultrasound and microbubble can enhance in vivo gene transfection efficiency in rats with injured achilles tendons and granulation tissues without causing obvious normal tissue damage, which could provide a sound basis for experimental study of gene therapy for injured tendons.