Objective To investigate the efficiency of target gene transfection of the heart and liver after tail vein or intramyocardial injection of adenovirus vector (GFP-Ad). Methods GFP-AD was constructed at first. A total of 20 male 8-week old C57BL/6 mice were randomly and equally divided into tail vein injection of GFP-AD group and intramyocardial injection of GFP-AD group. The mRNA levels of GFP in the heart and liver tissues were detected by Q-PCR at different time points. Fluorescence microscopy was performed to visualize the expression of GFP fluorescence. Results Compared with the tail vein injection group, the GFP mRNA level in mouse heart tissue was apparently higher in the intramyocardial injection group. In both groups, the GFP mRNA levels in liver tissue were significantly increased compared with that in the heart tissue. In the tail vein injection group, the GFP mRNA level in liver tissue reached a peak on day 7; but in the intramyocardial injection group, the mRNA level of GFP in liver tissue reached apeak on day 3. We also observed the same trend of GFP fluorescence expression in the tail vein injection group compared with that in the intramyocardial injection group.Conclusions Intramyocardial injection of adenovirus vector is suitable to achieve a higher transfection efficiency in mouse heart tissue compared with the tail vein injection method. Although both injection methods are suitable for transfection of mouse liver, the tail vein injection method is preferential for it is simple and less invasive.